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Roles of Dietary Calcium and Magnesium in Controlling Dairy Fecal Phosphorus Solubility

Permanent Link: http://ufdc.ufl.edu/UFE0021313/00001

Material Information

Title: Roles of Dietary Calcium and Magnesium in Controlling Dairy Fecal Phosphorus Solubility
Physical Description: 1 online resource (122 p.)
Language: english
Creator: Herrera, Daniel Antonio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: calcium, dairy, dietary, eutrophication, feces, manure, phosphorus, soils
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Soils receiving dairy manure (mixture of feces with urine) over long periods of time can be a non-point source of phosphorus (P) that potentially can degrade water quality. My research objectives were to evaluate the combined effect of physiological state and diet on dairy-feces P solubility and to test the hypothesis that fecal P solubility can be decreased by increasing available calcium (Ca) relative to magnesium (Mg) in the diet of lactating dairy cows. Feed and fecal samples were collected from animals in different physiological states and experimental multiparous cows (24) were fed eight diets with a 2x2x2 factorial arrangement involving two dietary Ca sources, two dietary Ca and Mg concentrations. Feed and fecal samples were collected and chemically analyzed for nutritive parameters, total and water-extractable P, Ca and Mg. The combined effect of physiological state and diet yielded fecal samples with different water-extractable P (WEP), despite comparable dietary P concentrations. Dietary treatments had little practical effects on animal performance parameters, digestibility of nutrients, and overall P balance of lactating dairy cows; however addition of CaCl2 had a tendency to decrease dry matter intake (DMI). Fecal samples with higher Ca and Mg concentrations showed reduced WEP; possibly, high Ca and Mg concentrations mutually suppressed dissolution of Ca,Mg-P forms by the common ion effect. This finding was supported by consecutive extraction data, SEM solid-state analysis and by XRD results from ashed fecal samples where hydroxyapatite (HAP), HAP plus Ca,Mg-P, and Ca,Mg-P were the P forms found in ashed fecal samples for high, intermediate, and low dietary available Ca, respectively. Increasing Ca concentration in the diet of lactating dairy cows preemptively reduced P release from incubated feces-soil mixtures. This effect was most pronounced in soils with low P retention capacity. No further P stabilization effect of high available dietary Ca was observed over a 42 week period in soil-feces incubations. This lack of a time effect suggests that Ca, Mg, and P interactions in the gastro intestinal tract (GIT) may be the major determinant of the subsequent environmental fate of fecal P; formation of stable Ca-P forms are determined in the GIT and changes in P forms upon application of feces to soils are unlikely.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Daniel Antonio Herrera.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Harris, Willie G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021313:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021313/00001

Material Information

Title: Roles of Dietary Calcium and Magnesium in Controlling Dairy Fecal Phosphorus Solubility
Physical Description: 1 online resource (122 p.)
Language: english
Creator: Herrera, Daniel Antonio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: calcium, dairy, dietary, eutrophication, feces, manure, phosphorus, soils
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Soils receiving dairy manure (mixture of feces with urine) over long periods of time can be a non-point source of phosphorus (P) that potentially can degrade water quality. My research objectives were to evaluate the combined effect of physiological state and diet on dairy-feces P solubility and to test the hypothesis that fecal P solubility can be decreased by increasing available calcium (Ca) relative to magnesium (Mg) in the diet of lactating dairy cows. Feed and fecal samples were collected from animals in different physiological states and experimental multiparous cows (24) were fed eight diets with a 2x2x2 factorial arrangement involving two dietary Ca sources, two dietary Ca and Mg concentrations. Feed and fecal samples were collected and chemically analyzed for nutritive parameters, total and water-extractable P, Ca and Mg. The combined effect of physiological state and diet yielded fecal samples with different water-extractable P (WEP), despite comparable dietary P concentrations. Dietary treatments had little practical effects on animal performance parameters, digestibility of nutrients, and overall P balance of lactating dairy cows; however addition of CaCl2 had a tendency to decrease dry matter intake (DMI). Fecal samples with higher Ca and Mg concentrations showed reduced WEP; possibly, high Ca and Mg concentrations mutually suppressed dissolution of Ca,Mg-P forms by the common ion effect. This finding was supported by consecutive extraction data, SEM solid-state analysis and by XRD results from ashed fecal samples where hydroxyapatite (HAP), HAP plus Ca,Mg-P, and Ca,Mg-P were the P forms found in ashed fecal samples for high, intermediate, and low dietary available Ca, respectively. Increasing Ca concentration in the diet of lactating dairy cows preemptively reduced P release from incubated feces-soil mixtures. This effect was most pronounced in soils with low P retention capacity. No further P stabilization effect of high available dietary Ca was observed over a 42 week period in soil-feces incubations. This lack of a time effect suggests that Ca, Mg, and P interactions in the gastro intestinal tract (GIT) may be the major determinant of the subsequent environmental fate of fecal P; formation of stable Ca-P forms are determined in the GIT and changes in P forms upon application of feces to soils are unlikely.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Daniel Antonio Herrera.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Harris, Willie G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021313:00001


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1 ROLES OF DIETARY CALCIUM AND MAGN ESIUM IN CONTROLLING DAIRY FECAL PHOSPHORUS SOLUBILITY By DANIEL A. HERRERA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Daniel A. Herrera

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3 A mi familia. My parents are an example of hard work, I am always amazed of their vision and effort to provide their kids with the tool s to succeed in life, particularly their encouragement to study and be the best we can possibly be. My siblings were like parents to me for most of my life, most of all, they are my friends and th ey know how much their children mean to me. I always felt their full support, particularly in di fficult times such as during my knee surgeries. My nieces and nephews were always the best excuse to go hom e. This dissertation is for you, my success is yours!

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4 ACKNOWLEDGMENTS I must acknowledge my committee members. Their leadership, knowledge, patience and charisma helped me in many ways. Dr. Willie Harris always encouraged me to go an extra step; he made my research project fe el like a true PhD learning experience. Dr. Charles Staples was extremely helpful in the animal side of my res earch and along with Dr. Jerry Sartain, made me work harder on statistics. Dr. Vimala Nair and Dr. Tom Obreza made strong contributions to my research design and planning. I thank all of them. I thank La Familia for their companionshi p, care, cooking style and uniqueness. Four years is a long time to live with me! They b ecame part of my personal life and were always willing to help with my research, including trips to feed the cows, collect samples and have a good time! I thank Rodrigo, Gerardo, Mario, Santia go, and Franklin for their help. I also thank rest of the Tico group for their friendship. I thank those who were involved in the comple tion of this work; Josan, Rocky, Lisa, Eric, Sergji, and the DRU personnel. All of them worked hard helping me, at some point they made my project theirs. Last, I thank Katya for being the special person she is. You became an inspiration to finish this work and I hope this is the be ginning of a long journey together.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION..................................................................................................................14 2 PHOSPHORUS RELEASE FROM DAIRY HEIFER AND COW FECES INFLUENCED BY PHYSIO LOGICAL STATE AND DIET..............................................20 Abstract....................................................................................................................... ............20 Introduction................................................................................................................... ..........20 Materials and Methods.......................................................................................................... .22 Results and Discussion......................................................................................................... ..24 Summary and Conclusions.....................................................................................................29 3 EFFECT OF DIETARY MODIFICATION S OF CALCIUM AND MAGNESIUM ON PERFORMANCE OF LACTATING DAIRY COWS...........................................................37 Introduction................................................................................................................... ..........37 Materials and Methods.......................................................................................................... .38 Cows, Diets, and Facilities..............................................................................................38 Sample Collection and Analysis......................................................................................40 Statistical Analysis..........................................................................................................43 Results and Discussion......................................................................................................... ..44 Diet Composition and Intake...........................................................................................44 Milk Production Parameters............................................................................................46 Nutrient Digestibility.......................................................................................................47 Balance of P, Ca and Mg.................................................................................................50 Conclusions.................................................................................................................... .........53 4 DIETARY CONTROL OF CALCIUM AN D MAGNESIUM AS A MEANS OF REDUCING PHOSPHORUS SOLU BILITY IN DAIRY FECES........................................69 Abstract....................................................................................................................... ............69 Introduction................................................................................................................... ..........69 Materials and Methods.......................................................................................................... .72 Results and Discussion......................................................................................................... ..76 Dietary and Total Fecal Concentrations of Phosphorus, Calcium, and Magnesium.......76

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6 Effect of Dietary and Fecal Concentrations of Ca and Mg on Water Extractable P, Ca, and Mg...................................................................................................................78 Relationship between Fecal WEP with WEMg and WECa Concentrations in Fecal Samples........................................................................................................................79 Impact of Dietary Modifications on Fecal-P Solubility..................................................82 Conclusions.................................................................................................................... .........83 5 DIETARY CALCIUM AND MAGNESIUM EFFECT ON PHOSPHORUS SOLUBILITY OF DAIRY FECES IN THREE DIFFERENT SOILS; AN INCUBATION STUDY.........................................................................................................93 Introduction................................................................................................................... ..........93 Materials and Methods.......................................................................................................... .94 Results and Discussion......................................................................................................... ..97 Conclusions.................................................................................................................... .......102 6 SUMMMARY AND CONCLUSIONS...............................................................................109 LIST OF REFERENCES.............................................................................................................113 BIOGRAPHICAL SKETCH.......................................................................................................122

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7 LIST OF TABLES Table page 2-1 Body weight, dry matter intake (DMI) a nd milk yield of three physiological groups: heifers, pregnant nonlactating (clo se-up), and lactating animals.......................................30 2-2 Ingredient and nutrient content of diets fe d to the three physiologi cal groups: heifers, pregnant nonlactating (close-u p), and lactating animals....................................................31 2-3 Cumulative water extractable (WE) P, Ca and Mg after 10 successive extractions in feces from Holstein heifers, pregnant nonlactating cows (close-up), and lactating cows........................................................................................................................... ........32 3-1 Ingredients and their concentration in dr y matter basis used in formulation of diets fed to lactating dairy cows.................................................................................................57 3-2 Chemical composition of diets fed to lactating dairy cows...............................................58 3-2 Least squared means and standard e rror of the mean for animal production parameters of lactating dairy cows receivi ng two Ca sources, two Ca concentrations, and two Mg concentrations................................................................................................59 3-3 P values for animal production parameters of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations............................................60 3-4 Least squared means and standard error of the mean for nutri ent digestibility of lactating dairy cows receiving two Ca s ources, two Ca concentrations, and two Mg concentrations................................................................................................................. ...61 3-5 P values for nutrient digestibility of lact ating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations..........................................................62 3-6 Least squared means and standard error of the mean for dry matter and water intake, urinary and fecal output a nd overall P retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concen trations, and two Mg concentrations................63 3-7 P values for dry matter and water intake, urinary and fecal output and overall P retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations.......................................................................64 3-8 Least squared means and standard error of the mean for dry matter and water intake, urinary and fecal output and overall Ca retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concen trations, and two Mg concentrations................65 3-9 P values for dry matter and water intake, urinary and fecal output and overall Ca retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations.......................................................................66

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8 3-10 Least squared means and SEM for dry ma tter and water intake, urinary and fecal output and overall Mg retention (g d-1) of lactating dairy co ws receiving two Ca sources, two Ca concentrations, and two Mg concentrations............................................67 3-11 P values for dry matter and water intake, urinary and fecal output and overall Mg retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations.......................................................................68 4-1 Nutrient content of diets with different Ca and Mg c oncentrations and two calcium sources fed to the lactating dairy cows..............................................................................84 4-2 P value for orthogonal single degree of freedom contrast for total dietary concentrations of phosphorus (P), calcium (Ca), and magnesium.....................................85 4-3 Total (T) fecal and waterextractable (WE) concentrati ons of P, Ca, and Mg in lactating dairy cows feces after ten successive extractions with water.............................85 4-4 P values for total (T), cumulative water-extractable (WE), and waterextractable:Total concentration of phosphor us (P), calcium (Ca), and magnesium (Mg) in feces of lactating dairy co ws receiving two Ca sources, two Ca concentrations, and two Mg concentrations.......................................................................86 4-5 Correlation coefficient (r2) between water-extr actable P (WEP) and water-extractable Ca (WECa), water-extractable Mg (WEM g) or water-extractable Ca+ waterextractable Mg in fecal samples from lact ating dairy cows fed diets differing in Ca source, Ca concentration and Mg concentration................................................................86 4-6 Least squared means and SEM for fecal output, P concentration in the feces, P excretion via feces and the cumulative WEP per kilogram of feces, per day per cow and its implications in a 1000 head farm of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations............................................87 4-7 P values for fecal output, P concentration in the feces, P excretion via feces and the cumulative water-extractable P per kilogr am of feces, per day per cow and its implications in a 1000 head fa rm of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations..........................................................88 5-1 Morphological summary of soils from wh ich surface horizon samples were collected for the incubation experiment..........................................................................................103 5-2 Chemical and physical characteristics of the surface horizon (Ap) from each soil type used for the incubation.....................................................................................................103 5-3 Chemical characteristics of fecal samples used for the incubation with three different soils orders................................................................................................................... ....104 5-4 ANOVA table of water-extractable P (WEP ) from soil-feces at different times of incubation (1, 25, and 42 weeks).....................................................................................104

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9 5-5 Water extractable P from a single extracti on at 1:100 soil-feces mix to water ratio at different times of incubation and the respect ive standard error of the mean (SEM).......105 5-6 ANOVA table of cumulative water-extractabl e P (WEP) after eight extractions of at 1:100 soil-feces to water ratio after 25 weeks of incubation...........................................105 5-7 Cumulative water extractable P (WEP) fr om ten consecutive extractions at 1:100 soil-feces mix to water ratio after six months of incubation and the respective standard error of the mean (SEM)...................................................................................105 5-8 P values of water extractable P (WEP) fo r single degree of freedom contrasts based on dietary variables from where fecal samples were collected........................................106

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10 LIST OF FIGURES Figure page 2-1 Concentration of P (a), Ca (b), and Mg (c ) in diet (% of diet dry matter) and feces (+ standard error bars) of heifers, pregnant nonlactating (close-up) and lactating dairy cattle. Columns with different letter superscr ipts within an element were different ( P < 0.05)........................................................................................................................ ........33 2-2 Cumulative water extractable P (WEP) concentrations (mg kg-1) (SD) with sequential extractions of feces from dair y animals in three physiological states; heifers, pregnant nonlactating (close-up) and lactating. Asteris (*) indicates differences ( P < 0.05) in cumulative WEP among physiological states............................34 2-3 Relationship between (a) water-extr actable P (WEP)and WECa+ WeMg over 10 extractions in feces of Holstein dairy anim als in three different physiological states and (b) proportion of fecal TP that was water-extractable (WEP:TP) with total Ca and Mg content in feces of Holstein animals. Circled data points represent those of the heifer group............................................................................................................... ...35 2-4 Relationship between water extractable P a nd water extractable Ca or Mg from feces of Holstein dairy animals in three differ ent physiological states (a) heifers, (b) pregnant nonlactating (close -up) and (c) lactating.............................................................36 3-1 Interaction effect of Ca by Mg dietar y concentrations in a) milk yield, b) body weight, and c) body weight change....................................................................................55 3-2 Interaction effect of a) Ca source by Ca concentration on urine pH and Ca source by Mg concentration on b) acid detergent fibe r (ADF) digestibility, and c) Mg balance (Mg retained).................................................................................................................. ...56 4-1 Variation in water extractable P with respect to changes in dietary and fecal concentrations of Mg and Ca.............................................................................................89 4-2 Cumulative water extractable P, Ca and Mg with changes in dietary concentrations of Ca and Mg................................................................................................................... ..90 4-3 X-ray diffraction analysis of ashed fecal samples of randomly selected dairy cows receiving (1) LoCaCO3, HiMg; (2) LoCaCO3, LoMg; (3) HiCaCO3, HiMg; and (4) HiCaCl2, HiMg. Minerals present are: C: calcite (CaCO3); S: silvite (KCl); W: whitlockite (Ca,Mg)3(PO4)2; and A: hydroxyl-apatite Ca5(PO4)3(OH, Cl, F). LoCa refers to low dietary Ca concentra tion whereas HiCa is high dietary Ca concentration; same applies to Mg. Each line within a graph re presents a different fecal sample................................................................................................................... ....91 4-4 Dot maps and P, Ca and Mg elementa l spectra from areas identified with high P concentrations of randomly selected fecal samples of cows being fed low Ca and high Mg concentrations or high Ca and high Mg concentration in their diets..................92

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11 5-1 Interaction effect of soil type and time on water-extractable P (WEP) single extractions at 1:100 water to soil-feces in cubations. Data points are averaged across diets.......................................................................................................................... ........107 5-2 Averaged water-extractable P (WEP) at three different times (1, 25, and 42 weeks) from single extractions at 1:100 ratio of water to soil-feces incubations........................107 5-3 Relationship between water-extractable P (WEP) with WECa+WEMg from eight consecutive extractions at 1:100 soil-feces to water ratio. Fecal samples were incubated for 25 weeks with a) Ultisol, b) Entisol, and c) Spodosol. Data points shown are average of four repl ications standard error..................................................108

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12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLES OF DIETARY CALCIUM AND MAGN ESIUM IN CONTROLLING DAIRY FECAL PHOSPHORUS SOLUBILITY By Daniel A. Herrera August 2007 Chair: Willie Harris Major: Soil and Water Science Soils receiving dairy manure (mixture of feces with urine) over long periods of time can be a non-point source of phosphorus (P ) that potentially can degrade water quality. My research objectives were to evaluate the combined effect of physiological state and diet on dairy-feces P solubility and to test the hypothesis that fecal P solubility can be decreased by increasing available calcium (Ca) relative to magnesium (Mg) in the diet of lactat ing dairy cows. Feed and fecal samples were collected from animals in different physiological states and experimental multiparous cows (24) were fed eight diets wi th a 2x2x2 factorial arra ngement involving two dietary Ca sources, two dietar y Ca and Mg concentrations. Feed and fecal samples were collected and chemically analyzed for nutritive pa rameters, total and water-extractable P, Ca and Mg. The combined effect of physiological state and diet yielded fecal samples with different water-extractable P (WEP), despit e comparable dietary P concentr ations. Dietary treatments had little practical effects on animal performance parameters, digestib ility of nutrients, and overall P balance of lactating dairy co ws; however addition of CaCl2 had a tendency to decrease dry matter intake (DMI). Fecal samples with highe r Ca and Mg concentrations showed reduced WEP; possibly, high Ca and Mg concentrations mutually suppressed di ssolution of Ca,Mg-P forms by the common ion effect. This finding was supported by consecutive extraction data,

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13 SEM solid-state analysis and by XRD results fr om ashed fecal samples where hydroxyapatite (HAP), HAP plus Ca,Mg-P, and Ca,Mg-P were the P forms found in ashed fecal samples for high, intermediate, and low dietary available Ca, respectively. Incr easing Ca concentration in the diet of lactating dairy cows preemptively reduce d P release from incubated feces-soil mixtures. This effect was most pronounced in soils with lo w P retention capacity. No further P stabilization effect of high available dietary Ca was observed over a 42 week period in soil-feces incubations. This lack of a time effect suggests that Ca, Mg, an d P interactions in the gastro intestinal tract (GIT) may be the major determinant of the subse quent environmental fate of fecal P; formation of stable Ca-P forms are determined in the GIT and changes in P forms upon application of feces to soils are unlikely.

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14 CHAPTER 1 INTRODUCTION Phosphorus (P) is an essential element for liv ing organisms. Increased P concentration in diets of lactating dairy cows has been shown to positively affect feed intake, milk production (Call et al., 1987), and reproducti ve performance (Scharp, 1979, St eevens et al., 1971). This perception of improved economical benefits associ ated with increased P feeding has led farmers to overfeed P to dairy cattle (T allam et al., 2005). However, f eeding P in excess of National Research Council (NRC) (2001) re commendations has not shown evidence of improved animal performance over a 2 year pe riod (Wu and Satter, 2000). Intensive dairy production often re quires import of feedstuffs to meet nutrient requirements of animals and maximize milk production. Import of feedstuffs creates a nutrient imbalances for the farm system; nutrient inputs (feed) exceed ou tputs (milk and meat). Up to 72% of the P imported can accumulate on the farm (Klausner et al., 1998). Manure from these operations is the end point where excess nutrients are collected. This manure is frequently applied to soil in spray fields. High intensity areas (HIA) close to areas where animals queue for milking receive high fecal loading as well. An increase in soil P (soil test P) con centration has been documented where manure applications to soils have exceeded crop removal for long periods of time (Daniel and Lemunyon, 1998; Sharpley, 1996; Sims et al., 2000; Whalen and Chang, 2001). Soils receiving dairy manure can become a source of P for offs ite movement. This P ma y cause eutrophication, which can eventually lead to a decrease in th e aesthetic and recreationa l value of water bodies (Ebeling et al., 2002; Sharpley et al., 1994). Phosphorus movement from agricultural areas has become an increasingly environmental concern. According to the United States Environmental

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15 Protection Agency (USEPA, 2000), agriculture is the leading source of water quality impairments of rivers an d lakes in the USA. Application of manure to crops based on P c oncentrations is an approach that can minimize P buildup in soils around confined anim al feeding operations, but this approach restricts manure application areas or increases costs due to transporta tion (Dou et al., 2003). Another research focus to optimize P balance in farms is to decrease P in animal diets and therefore P concentration in f eces, the main route of P excre tion (Morse et al., 1992). Lowering dietary P has successfully decrease d fecal-P concentrations as diet ary P concentration is one of the dominating factors affecting fecal P excretion (Chapuis-Lardy et al., 2004; Toor et al., 2005a; Wu et al., 2001). Dietary modifi cations have reduced fecal P concentrations up to 33% when used in commercial dairy farms (Cerosaletti et al., 2004), particularly decreasing the watersoluble P fraction (Dou et al., 2002 ) with no detrimental effects on animal productivity (Wu and Satter, 2000). There is a limit to which dietary and ther efore manure P can be reduced because dairy cows have a threshold for P excretion. Base d on NRC recommendations P should be fed to lactating dairy cows at approximately 3.7 g kg-1 of dietary DM (NRC, 2001). Contrasting findings on animal performance have been report ed when feeding P at concentrations below NRC recommendations. In diffe rent experiments; Wu et al., (2000), documented negative impacts on animal performance when feeding 3.1 g of P kg-1 of dietary DM, whereas Dou et al. (2002) concluded that 3.1 to 3.7 g of P kg-1 of dietary DM was adequa te or near adequate for milk production. Irrespective of dietary P concentr ation, fecal P concentrations remained above 4.0 g kg-1 of feces DM even when cows we re fed diets containing P at 3.1 g kg-1 of dietary DM (Wu et al., 2000).

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16 Differences in concentrations and release char acteristics of P in ma nure, independent of dietary P intake, also have been documented wh en comparing animals of different type or physiological state (Nair et al ., 2003). Therefore, both animal requirement and dietary P concentration are relevant factors that should be accounted for when considering P excretion via feces. To optimize P balance in farms, emphasis should be placed not only on reducing dietary and fecal P concentrations but also on P stabil ity in the long term. Feces-derived components can play an important role in the fate of P once appl ied to the soil, particularly in soils with low P sorbing capacity (Josan et al., 2005). Interactions between Ca and Mg with P can play a major role in P nutrition and therefore excretion. Dietary concentrations of Ca and Mg, number of days of lactation and manure pH have been shown to influence excretion and solu bility of fecal P from ruminants (Chapuis-Lardy et al., 2004; Nair et al., 2003). Ruminants maintain Ca homeostasis with rema rkable precision, particularly their blood concentration because of the importance of Ca in muscle contraction (heart) and nerve transmission. Contrary to Ca, ho meostasis control of P and Mg is less rigorous. Interactions between Ca, Mg and P in combination with the effect of parathyroid hormone, calcitonin and vitamin D play an important role in homeostasis of Ca, P, and Mg; for in stance, excessive dietary Ca can negatively impact Mg and P absorption (Reinhardt et al., 1988) and Cu liver storage (Huber and Price, 1971). Adequate di etary concentrations of Ca and P, their chemical forms, and ratio of Ca:P are the primary factors influenci ng their absorption. A dietary Ca:P ratio between 1:1 and 2:1 is usually recommende d; however, ruminants can tolerate a wider range, particularly when the animal has adequate supp ly of vitamin D (McDowell, 2003).

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17 Metabolism of Ca, Mg and P in domestic anim als is extremely complex (Littledike and Goff, 1987). Calcium absorption is highly regulat ed in ruminants (with exception of aged lactating cows). Homeostasis mechanisms allo w for a certain degree of dietary manipulation without negatively impacting animal heath. Alth ough dietary Ca:P ratios greater than 2:1 are above NCR (2001) recommendations, they are often fed to lactating dairy cows in research experiments (Dann et al., 2006) and are a common practice in rati ons for pregnant non-lactating pregnant cows (far-off or clos e-up) (Chan et al., 2006, Tucker et al., 1991) with no evidence of negative effects on P metabolism. Diets offered to dairy cows in the last da ys of pregnancy (close-up animals) can be formulated to contain additional anions such as Cl and S. These are referred to as negative DCAD diets (dietary cation-anion difference) based on the equation (Na+ K-Cl-S). These diets are formulated to promote Ca mobilization from b one so as to reduce the incidence of milk fever once the animal is in lactation. Feeding nega tive DCAD diets (-5 to -10 meq/100 g of DM) increased H+ fluxes as a companion cation upon higher ab sorption of Cl and S, causing mild metabolic acidosis. To buffer and maintain blood pH, the animal responds by increasing bone resorption, mobilizing bicarbonate; along with it, Ca is also mobilized and th erefore the animal is metabolically prepared to respond to the increased Ca demand from milk production (Chan et al., 2006, Goff and Horst, 1993, Goff et al., 2004, Tucker et al., 1991). Calcium chloride is one of the anionic salts used in form ulation of negative DCAD diets. While these diets are fed to animals in a specific physiological state and for shor t periods of time, they provide evidence that an increased dietary Ca:P ratio is not detr imental to animal health or performance. Lactating dairy cows in the southeastern Unite d States experience heat stress, particularly between June 1st and September 30th (West, 2003). Sweating and accel erated respiration are

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18 means utilized by ruminants to dissipate heat and maintain body temperature. These mechanisms increase the animals requirements for Na and K (Sanchez et al., 1994). Increased dietary K has a suppressant effect on Mg absorption which can lead to hypomagnesemia (grass tetany); however, it can be effectively counteracted with addi tional Mg supplementation (Jittakhot et al., 2004). The NRC (2001) recommends increased Mg concentra tion in diets of lacta ting dairy cows when fed high dietary K. The main drawback of incr eased dietary Mg is reduced dry matter intake (DMI), particularly above 4.0 g kg-1 of DM (Sanchez et al., 1994), ma inly as salts of sulfate and chloride. Ruminants can excrete large amounts of Mg via urine therefore Mg toxicity is not a practical problem in dairy cows (NRC, 2001). Although the small intestine is the main absorption site for Ca (Horst et al., 1994) and P (Grace et al., 1974) and the reticulo-rumen is for Mg (Tomas and Potter, 1976), interaction between these elements in the animal has been h ypothesized to play a role in the inorganic form in which P is excreted and therefore in its so lubility in manures of monogastric animals, particularly poultry manures (Cooperband a nd Good, 2002; Toor et al., 2005b); additionally, manures from turkey fed a Ca:P ratio above 2 experienced a transforma tion of more soluble (dicalcium phosphate) to less so luble P compounds (hydroxylapatit e) in their manure (Toor et al., 2005b). Manure-impacted soils often have conditions associated with increased P stability because dairy manure has (with respect to soils) elevated concentratio ns of P and Ca and high pH; however, no solid state crystalline phosphate minera ls have been identified in these soils (Harris et al., 1994) or in (dairy) manure-soil incubations (Cooperb and and Good, 2002). Instead, P remains highly soluble, even afte r years of abandonment (Josan et al., 2005; Nair et al., 1995). High concentrations of Mg a nd dissolved organic carbon have been proposed as potential

PAGE 19

19 inhibitors of more stable calcium-phosphate mine rals (Ca-P) in manure-amended soils (Harris et al., 1994; Josan et al., 2005). From an animal nutrition standpoint, large intake s of Ca and Mg are of concern because of their effect on decreasing P ab sorption by the formation of inso luble phosphates in the gastro intestinal tract (GIT) (McDowell, 2003). However from an environmental perspective and given the importance on reducing P losses from agricu ltural areas (Sims et al., 2000; USEPA, 2000), interactions of Ca and Mg with P to decrease P solubility in dairy manures is a desired characteristic. Thus, the question before us is: Ca n dietary Ca be increased to reduce P solubility in feces from lactating dairy co ws without negatively impacting animal health and performance? My study tests the following hypotheses: H1: The combination of physiological state of the dairy cow and diet found on dairy farms results in feces with different P release characteristics. H2: Higher availability of dietary Ca relative to Mg can decrease fecal P solubility by favoring formation of more stable calcium phosphates, with no detrimental effects on animal health and performance.

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20 CHAPTER 2 PHOSPHORUS RELEASE FROM DAIRY HEIFER AND COW FECES INFLUENCED BY PHYSIOLOGICAL STATE AND DIET Abstract The objective of this study was to evaluate the combined effe ct of diet and physiological state of Holstein animals on concentration and solubility of P, Ca, and Mg in feces, using standard diets of comparable P concentration. Diet ary ingredients and fecal samples from heifers, pregnant, nonlactating cows within three week s of calving; and lacta ting dairy cows were collected and analyzed for Ca, Mg, and P concentr ation. Solubility of th ese three minerals in feces was determined using repeated water extr actions at a 1:50 feces:w ater ratio. Increased dietary concentrations of P, Ca and Mg did not result in increased total concentrations of those nutrients in feces among physiologica l states. Total P (TP) concentra tion in feces did not relate to water-extractable P (WEP), but WE P differed among physiological groups ( P < 0.006). Of the three groups, feces from heifers had the highest P solubility despite having the lowest TP fecal concentrations. Increased ratio of P:(Ca+Mg) resu lted in increased WEP. Release of P from fecal samples was highly associated with release of Ca and Mg; the correlation coefficient was greater for Mg than Ca. Total concentration of Ca and Mg in feces as well as the form in which those nutrients are present may be important when c onsidering potential for off-site P movement. Feeding dairy animals close to their requirement s reduces both total and particularly waterextractable P concentrations in fecal samples. Introduction Phosphorus (P) movement from agricultu ral areas has increasingly become an environmental concern. According to United St ates Environmental Protection Agency (USEPA, 2000), agriculture is the leading s ource of water quality impairment s in rivers and lakes in the USA. Intensive dairy production re quires import of feedstuffs to complement those produced on

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21 the farm, if any, to meet nutrient requirements of animals and maximize milk production. With the import of feedstuffs into the system, a nutri ent imbalance is created on farm where nutrient inputs (feed) exceed outputs (milk and meat). Up to 72% of the P imported can accumulate on the farm (Klausner et al., 1998). Manure from these operations is the end point where excess nutrients are collected. An increase in soil P (s oil test P) concentration has been documented where manure applications to soils have exceed ed crop removal for long periods of time (Daniel and Lemunyon, 1998; Sharpley, 1996; Sims et al., 2000; Whalen and Chang, 2001). Soils receiving dairy manure can become a source of P for offsite movement. This P may cause eutrophication, which can eventually lead to a decrease in the aesth etic and recreat ional value of water bodies (Ebeling et al., 2002; Sharpley et al., 1994). To minimize P release from dairymanure-im pacted soils, a common approach has been to decrease dietary concentrations of P fed to dair y cows (Cerosaletti et al ., 2004; Dou et al., 2003). Lowering P in the diet results in decreased fecal-P concentrations as dietary P concentration is one of the dominating factors affecting fecal P ex cretion (Chapuis-Lardy et al., 2004; Toor et al., 2005; Wu et al., 2001). However, this approa ch has a baseline limit below which animal performance is negatively affected; feeding P belo w that point is questionable, as pointed out by Chapuis-Lardy et al. (2004). Diffe rences in P concentrations and P release characteristics of manure, independent of dietary P intake, also have been documented when comparing animals of different type or physiological st ate. Dietary concentrations of Ca and Mg, days in milk and manure pH also have been shown to influence ex cretion and solubility of fecal P (Chapuis-Lardy et al., 2004; Nair et al., 2003). Recent studies hypothesized that Mg in feces, in addition to Ca, may play a role in promoting P solubility in manure-amended soils due to the greater solubility of Mg phosphates

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22 relative to Ca phosphates (Josan et al; 2005; Nair et al., 2003). The potential influence of Mg may be dictated ultimately by diet ary factors. Diets routinely form ulated for animals in different physiological states contain varyi ng concentrations of Mg and Ca, which could affect P solubility in feces regardless of total P in the diet. Objectives of this study were (i) to compare P solubility of dairy feces from animals in different physiolo gical states consuming diets tailored to their respective needs and (ii) to relate P rel ease from feces to Ca and Mg release. Materials and Methods Five Holstein animals in each of three physio logically different groups heifers, close-up cows (pregnant, nonlactating dair y cows within three weeks of calving) and lactating cows were used at the University of Florida Dairy Res earch Unit (DRU). The heifer group consisted of animals between 14-16 months of age and fed to gain 0.9 kg d-1. Lactating group was composed of three multiparous and two primiparous cows, w ith 13 ( 8) days in milk; close-up group had two multiparous and three primiparous animals a nd averaged 260 ( 10) days of gestation; detailed information on animals is given in Table 2-1. Ingredient and chemical composition of diets fed to each group (Table 2-2) represent those used by commercial dairy farms in Florida. Di ets differed in P, Ca and Mg concentrations because they were formulated to meet requiremen ts of animals in different physiological states. The diet for the close-up group was formulated to contain more Ca, Mg, S, and Cl in order to establish metabolic acidosis and reduce the risk of paripa rturient paresis. All ingredients, except forages (Table 2-2) we re mixed together to form a concentrate mix. The concentrate mix and forages were then mixed and fed as a total mixed ration daily. Individual samples of forages and concentrate mixes were collected 3 d prior to the days of fecal collection and composited. Compos ited and dried feed samples were analyzed for DM (105C for 8 h), neutral detergent fiber (NDF) using heat-stable -amylase (Goering and Van Soest,

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23 1970; Van Soest et al., 1991), acid detergent fi ber (ADF) (AOAC, 1990), and total nitrogen (N); crude protein (CP) was calculated by multiply ing N x 6.25(Elementar Analysensysteme, Hanau, Germany). Crude protein was calculated by multip lying N x 6.25. In addition, a composites were sent to a DHIA Forage Testing Laboratory (D airy One, Ithaca, NY) where samples were analyzed by wet chemistry for Ca, P, Mg, K, Na, Zn, Cu, Mn, and Fe by the ignition method (Andersen, 1976); Cl was determined by titration with AgNO3 using Brinkman Metrohm 716 Titrino Titration Unit with silver electrode (Metrohm Ltd., C-H-9101 Herisau, Switzerland) and S by oxidation (Leco Model SC432, Leco Instruments, Inc). Animals were accustomed to each diet for at l east 10 d prior to fecal collection. To obtain a composited fecal sample per animal, approximately 1000 g of wet feces were collected twice in 1 d from the rectum of each of five animals in the three physiological groups. Fecal samples were dried at 55C in a forced-air oven and ground to pass the 2-mm screen of a Wiley mill (A.H. Thomas, Philadelphia, PA). Fecal samples were analyzed in quadruplicate for total P, Ca and Mg by the ignition method (Andersen, 1976). Calcium and Mg were m easured by atomic absorption spectroscopy; P was determined on a UV-visible recording spec trophotometer at 880 nm wave-length via the molybdate-blue colorimetric me thod (Murphy and Riley, 1962). Dried fecal samples were mixed with distilled water at a ratio of 1: 50 g of DM:mL, shaken for 1 h, and centrifuged at 1000x g for 5 min. The supernatants were collected and filtered through a 0.45 m filter. Ten successive extractions were carried out at room temperature (~25 C). Supernatant solutions were analyzed for soluble reactive P, Ca, and Mg, using the same methods specified above for total determina tion of these elements in fecal samples.

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24 Fecal samples were dried to standardize all sa mples to the same DM content so that the same amount of moisture+DM from each fecal sample would be mixed with distilled water for extraction. Drying is also effectiv e to obtain consistency in sample treatment, preserve samples, and avoid DM heterogeneity in feces between an imals and within animals in different sampling events. Furthermore, feces (or manure) often und ergo a drying period upon application to soil. We are aware of the possible effects on P sol ubility when fecal samples are dried. Previous research has reported contradict ory findings, Ajiboye et al. ( 2000) reported an increased in water-extractable P (WEP) from dairy cow feces when samples were oven-dried at 105 C whereas Chapuis-Lardy et al. ( 2004) showed a decrease in inorga nic P soluble in water when dairy cow fecal samples were dried at 65 C. We believe that relative treatment effects would not be compromised by drying at low te mperature, as Dou et al. (2000) reported that ~70% of P in dairy cow manure was water soluble and most of it was extracted in the initial steps of repeated water extractions. Statistical Analyses. Single data measurements of Ca, Mg and P were analyzed using the GLM procedure of SAS whereas Mixed Linear M odel was used for repeated measures from consecutive extractions of the same elements; s lice option was used to detect differences in individual extractions. Treatment differences were evaluated using the F-protected least significant difference test. Correlations and nonl inear regressions were used to describe relationships between elements in our analysis. Di fferences discussed in th e text were significant at P 0.05 unless otherwise indicated. Results and Discussion Diets differed in nutrient concentrations sin ce each was customized to meet the nutrient requirements or to initiate a desired physiologi cal response of animals at a given physiological state (Table 2-2). These dietary differences among groups of anim als were large for Ca and Mg,

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25 but minimal for P. Concentrations of P, Ca, and Mg were greater in feces than in the diet (Fig. 2-1) although no statistical analysis was perfor med for this comparison because feed samples were not replicated. Increased concentration of mine rals in feces as compared to the diet suggests greater digestibility of other f eed components, protein for exampl e, compared to that of the minerals measured. Although the concentrations of dietary P were similar for each animal group (Table 2-2), P concentration in fecal samples from close-up cows differed from that of lactating cows but P in fecal samples from heifers did not differed from a ny of the other two groups (Fig. 2-1). Dietary P requirements for heifers, clos e-up and lactating animals base d on NRC (2001) recommendations are 2.4, 3.0-4.0, and 3.7 g kg-1 of dietary DM respectively. Phos phorus concentration of diets fed in this study ranged from 3.4 3.7 g kg-1 of dietary DM (Table 2-2) Therefore the heifer group was fed a diet greater in P concentration (3.4 g kg-1) than recommended (2.4 g kg-1). The P concentration in the diet of close-up cows was in creased intentionally so that when feed intake decreases around the time of parturition, intake of P is adequate to meet tissue and fetal needs. However close-up cows in this study were several days away from parturit ion and therefore, feed intake had not decreased resulting in greater P inta ke than needed by tissues and fetus. Dietary P concentrations above animal requirements marked ly increased P fecal concentration as observed in feces from heifers (5.8 g kg-1) and close-up cows (6.4 g kg-1) (Fig. 2-1), suggesting that dietary P concentration is not the only factor affecting total fecal P concentration but also dietary concentration with respect to animal requirements. Increased fecal P concentration with increa sed concentration of dietary P has been documented widely when working with animals in the same physiological state (e.g. lactating dairy cows), particularly when animals were fe d above their P requirement. For example, Dou et

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26 al. (2002) reported that increasi ng dietary P concentrations positively correlated with higher P concentrations in feces; others have reported si milar results (Dou et al., 2003; Toor et al., 2005a; Valk et al., 2002; Wu et al., 2000). Results of the present study conf irm the importance of considering animal P requirements and P intake as factors affecting P conten t of feces. However, fecal P concentrations do not represent total ou tput of fecal P. Lactat ing cows fed diets of adequate P concentration (mg kg-1) may have greater total P output (g d-1) because of high feed consumption compared to heifers fed diet s containing somewhat excessive dietary P concentrations. Diets with increased Ca or Mg concentration did not coincide with increased Ca or Mg concentration in feces, respectivel y. Diet for close-up cows contai ned a greater Mg concentration than that of lactating cows (Table 2-2) whereas the opposite was true for fecal Mg concentrations (Fig. 2-1c). Heifers had the lowest Mg concentr ation in diet and feces; the same trend observed for dietary and fecal Mg concentrations among gr oups was also documented for Ca. In the case of Mg, the inorganic Mg supplement can be a factor influencing the results. Concentration of Mg in the mineral supplement of pregnant nonlactati ng and lactating cows was similar (3.0 and 2.9 g kg-1 respectively) but the source of Mg differed. Lactating cows were fed only MgO, whereas pregnant nonlactating cows were fed MgO and MgSO4. The absorption coefficient for Mg in MgO is 0.7 and in MgSO4 is 0.9 (NRC, 2001). Higher availability of Mg would mean higher absorption and therefore reduced f ecal excretion, helping to account for the fact that there was reduced Mg in feces of animals receiving MgSO4 in their diet. Heifers received no inorganic Mg supplement; an absorption coefficient of 0.16 was assigned for Mg from natural feedstuffs (NRC, 2001).

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27 Feces from Holstein females in different phys iological states not only differed in mineral concentration but also in water-extractable mi neral (Table 2-3). Water-extractable P made up 86% of the total fecal P of heifers but only 47 an d 42% of that of close-up and lactating cows, respectively (Table 2-3). Water-e xtractable Ca and Mg as a pr oportion of total Ca and Mg was lower for lactating cows compared with the othe r two groups (20% vs. ~42% for Ca and 56% vs. ~87% for Mg); indicating Ca is present in a less so luble form, consistent w ith the solubility of P from feces from lactating cows, suggesting an increas ed association of P with Ca rather than Mg. Absorption of P in the gastroin testinal tract of the animal is influenced by amount of P intake, source of P, intestinal pH, age of animal, intestinal para sitism, and intakes of Ca, Fe, Al, Mn, K and Mg (McDowell, 2003). Cumulative wate r-extractable P was weakly correlated with fecal P concentration (r2 = 0.22 P =0.08). Although different ratios were used in previous research, positive correlation between TP and WE P in manure has been reported (e. g., ChapuisLardy et al., 2004; Dou et al., 2002; Dou et al., 2003) For example He et al. (2004), using a 1:100 manure to water ratio, reported a r2=0.62 between H2O-extracted Pi and TP in manure. This relationship could be influenced by di etary concentration with respect to animal requirements. The heifer group was fed P above their requirements that can increase the water soluble P portion in manure as s hown previously by Dou et al. (2002). Fecal samples from three physiological groups differed in WEP at the first extraction and thereafter in cumulative WEP ( P < 0.05) (Fig. 2-2). Fecal samples from the heifer group had the greater WEP both in concentration and as percent of TP dissolved at every extraction. With three cumulative extractions over 50% of the total extracted P was solubilized. At this point WEP from fecal samples of heifers had solubilized 57% of the total dissolved compared with 53% for closeup and lactating cows. Three extractions can be considered enough to evaluate relative

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28 differences in WE minerals among fecal sample s; however, ten extractions were done as an indication of the long term solub ility of minerals. Feces with hi gh P concentration also had the greater WEP:TP ratio in feces (see heifers in Ta ble 2-3). For Ca and Mg however, increased total concentration in feces was not related to greater soluble proportion (Table 2-3). Decreased ratio of P:(Ca+Mg) in feces increas es the possibility of interaction of these cations with P; this interaction may result in a less soluble form of P in feces. This ratio was greatest in feces from heifers (0.31), followed by close-up cows (0.22) and lactating cows (0.10) (Fig. 2-1). The ratio of P:(Ca+Mg) in feces influe nced WEP. Fecal samples with the lowest ratio (from lactating cows) had the lowest concentration of WEP (1655 mg kg-1) (Table 2-3; Fig 2-1). These results are in agreement w ith previous reports where it ha s been suggested that increased Mg and Ca concentrations in manure and manureamended soils (Nair et al., 2003; Sharpley et al., 2004) could be the reason for the increase in either Mehlich-1 or Mehlich-3 extractable P in samples despite low WEP concentrations. They s uggested that the presence of Ca-P complexes was responsible for such behavior. This idea is c onsistent with results from our experiment when WEP was compared in a regression analysis to tota l concentrations of eith er Ca or Mg in feces (Fig. 2-3b). Concentrations of water-extractable Ca+Mg we re highly correlated w ith WEP in feces of Holstein dairy animals in three different physio logical states (Fig. 23a). Close association between these elements suggests that a Ca-P, Mg -P and/or Ca,Mg-P solid phase controls the initial release of P from feces. However, it is evident based on feces analysis by animal group that the relationship of water-extr actable Ca and Mg to WEP is not the same across fecal sources. To further explore the associati on of Ca and Mg with WEP, total Ca and Mg concentrations in feces were plotted against the fraction of fecal TP that was water extractable (WEP:TP) (Fig.

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29 2-3b). Increased Ca and Mg concen trations in feces were negativ ely correlated with WEP:TP, a relationship ( P < 0.001) best described by a power functi on. We recognize this evidence is not conclusive, as other forms of Ca (CaCO3 vs. CaCl2) and Mg (MgO vs. MgSO4) can influence the relationship of these two elements with P. Pronounced differences between heifers and adult animals (pregnant nonlactating and lactating) are apparent in th e relationship between WEP:TP ratio and total Ca or Mg (Fi g. 2-3b). The heifer group had the higher proportion of TP as WEP and clustered separately from th e other two groups. Data points fo r heifers are circled in Figure 2-3b. When heifer data were removed, increased tota l Ca or Mg concentrations in feces of adult animals was not associated with a distinct decrease in WEP:TP. Water extractable Ca and Mg showed a str ong power function relationship with WEP for all physiological states; however r2 values were consistently higher for WEMg than WECa across physiological states (Fig. 2-4 a, b and c). These finding s confirm a close association between P release and that of Ca and Mg, a nd suggest the presence of a Mg,Ca-P phase associated with P release from fecal samples. Similar results have been reported in dairy, layer chicken and swine manures (Kleinma n et al., 2005) and in dairy manure-impacted soils (Josan et al., 2005). Summary and Conclusions Total P fecal concentrations did not relate to P extracted after 10 consecutive water extractions. Calcium and Mg in feces seem to play an important role in controlling WEP. Increased ratio of P:(Ca+Mg) in feces corresponde d to increased WEP. The proportion of TP that was water-extractable also was reduced by increa sed Ca and Mg in feces, although this may have been influenced by the physiol ogical state of the animals. An imals in different physiological states and fed different diets produced feces with different P release characteristics. Release of P from feces as measured using successive water-ext ractions strongly related to rel ease of Mg and

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30 Ca, with the relation between Mg and P being st ronger. The proportion of TP solubilized after ten consecutive extractions went from ~42% for adult lactating cows fed diets matching their P requirements to ~86% for growing heifers fed a di et containing P at 140% of their requirement. Results of this study indicate that, dietary P concentration with respect to animal P requirements, and dietary and fecal concentrations of Ca and Mg influence P solubility in feces of Holstein dairy cattle. Table 2-1. Body weight, dry matte r intake (DMI) and milk yiel d of three physiological groups: heifers, pregnant nonlactating (clo se-up), and lactating animals. Body Weight DMI Milk Yield kg --kg day-1 --Heifers 380 () 10.2 (.5) -Close-up Primiparous 570 () 8.0 (.2) -Multiparous 723 () 11.0 (.5) -Lactating Primiparous 550 () 14 (.6) 26 (.7) Multiparous 680 () 16 (.8) 29 (.4)

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31 Table 2-2. Ingredient and nutrien t content of diets fed to the th ree physiological groups: heifers, pregnant nonlactating (close-u p), and lactating animals. Heifers Close-up Lactating Ingredient --------------g kg-1 ---------------Corn silage 363 450 375 Bermudagrass hay 300 150 -Alfalfa hay --100 Ground corn 41 146 220 Citrus pulp 131 50 50 Cottonseed hulls 87.0 -20.0 Minerals and vitamins premix 17.0 65.0 50.0 Extruded Soybean meal --70.0 Soybean meal 61.0 125.0 100.0 Sunflower oil -14.0 15.0 Chemical composition Crude Protein 105.0 134.0 151.0 Neutral Detergent Fiber 500.0 390.0 318.0 Acid Detergent Fiber 283.0 208.0 188.0 Ether extract 31.0 46.0 43.0 Ca 4.5 15.6 7.8 P 3.4 3.1 3.7 Mg 1.5 3.3 2.8 K 14.0 14.0 15.0 Na 1.5 2.2 5.0 S 3.1 4.2 2.3 Cl 4.6 8.1 4.4 Fe 0.15 0.3 0.2 Zn 0.04 0.04 0.1 Cu 0.01 0.02 0.02 Mn 0.04 0.03 0.07 Ingredients used for the Minerals and vitamins premix are specified; Heifers = Corn Meal, monocalcium phosphate, dicalcium phosphate, ammonium sulfate, salt, copper sulfate, sodium selenite, ethilenediamine dihydriodite, manganous sulfate, zinc sulfate, vitamin A supplement, vitamin E supplement, and stabilized feed fat. Pregnant nonlactating= Calcium carbonate, monocalcium phosphate, dicalcium phosphate, magnesium oxide, salt, potassium sulfate, magnesium sulfate, sodium selenite, cobalt sulfate, copper sulfate, zinc sulfate, manganous oxide, calcium iodate, am monium chloride, calcium sulfate, vitamin A supplement, vitamin D3 supplement, vitamin E supplement, rice mill bypro duct (vitamin carrier), a nd stabilized feed fat. Lactating= Calcium carbonate, sodium sesquicarbonate, urea, dicalcium phosphate, monocalcium phosphate, roughage products, processed grain byproducts, magnesium oxide, salt, magnesium sulfate, potassium sulfate, potassium chloride, niacin supplement, zinc sulfate, manganese sulfate, vitamin E supplement, ferrous sulfate, copper sulfate, vitamin A supple ment, cobalt carbonate, vitamin D3 supplement, ethilenediamine dihydriodite, sodium selenite.

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32 Table 2-3. Cumulative water extractable (WE) P, Ca, and Mg after 10 successive extractions in feces from Holstein heifers, pregnant nonlactating cows (close-up), and lactating cows. Heifers Close-up Lactating ------------mg kg-1 ------------P 5034a 2943b 1655c Ca 6289b 9110a 6583b Mg 3543b 5538a 4731a ------------WE mineral : Total Mineral ------------P 0.86a 0.47b 0.42b Ca 0.44a 0.40a 0.20b Mg 0.91a 0.84a 0.56b For each element, mean values within a row fo llowed by a different letter were significantly different ( P < 0.05) as determined by F-protected least significant difference test.

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33 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 HeiferClose-UpLactating Physiological StateP (g kg-1) Manure-P Diet-P 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 HeiferClose-UpLactating Physiological StateCa (g kg-1) Manure-Ca Diet-Ca c 0.0 2.0 4.0 6.0 8.0 10.0 HeiferClose-UpLactating Physiological StateMg (g kg-1) Feces Dieta b cAB A B C B A C B A 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 HeiferClose-UpLactating Physiological StateP (g kg-1) Manure-P Diet-P 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 HeiferClose-UpLactating Physiological StateCa (g kg-1) Manure-Ca Diet-Ca c 0.0 2.0 4.0 6.0 8.0 10.0 HeiferClose-UpLactating Physiological StateMg (g kg-1) Feces Dieta b cAB A B C B A C B A Figure 2-1. Concentration of P (a), Ca (b), and Mg (c) in diet (% of diet dry matte r) and feces (+ standard error bars) of heifers, pregnant nonlactating (close-up) and lactating dairy cattle. Columns with different letter superscr ipts within an element were different ( P < 0.05).

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34 0 1000 2000 3000 4000 5000 6000 012345678910 ExtractionWEP (mg kg-1) Heifers Close-up Lactating********** 0 1000 2000 3000 4000 5000 6000 012345678910 ExtractionWEP (mg kg-1) Heifers Close-up Lactating********** Figure 2-2. Cumulative water extracta ble P (WEP) concentrations (mg kg-1) (SD) with sequential extractions of feces from dair y animals in three physiological states; heifers, pregnant nonlactati ng (close-up), and lactating. Asteris (*) indicates differences ( P < 0.05) in cumulative WEP among physiological states.

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35 y = 12.7Ln(Ca+Mg) 25.0 r2 = 0.94 y = 5.7Ln(Ca+Mg) 9.4 r2 = 0.86 y = 4.3Ln(Ca+Mg) 8.3 r2 = 0.94 0 5 10 15 20 25 30 35 40 45 050100150200250300350 WECa+WEMg (mmol kg-1)WEP (mmol kg -1) Heifers Close-Up Lactating y = 287Ca-0.98 r2 = 0.80 y = 243.6 Mg-1.1 r2 = 0.84 0.00 0.20 0.40 0.60 0.80 1.00 1.20 02004006008001,0001,200 Total Ca, Mg (mmol kg-1)WEP TP-1 Mg Ca a b y = 12.7Ln(Ca+Mg) 25.0 r2 = 0.94 y = 5.7Ln(Ca+Mg) 9.4 r2 = 0.86 y = 4.3Ln(Ca+Mg) 8.3 r2 = 0.94 0 5 10 15 20 25 30 35 40 45 050100150200250300350 WECa+WEMg (mmol kg-1)WEP (mmol kg -1) Heifers Close-Up Lactating y = 287Ca-0.98 r2 = 0.80 y = 243.6 Mg-1.1 r2 = 0.84 0.00 0.20 0.40 0.60 0.80 1.00 1.20 02004006008001,0001,200 Total Ca, Mg (mmol kg-1)WEP TP-1 Mg Ca y = 287Ca-0.98 r2 = 0.80 y = 243.6 Mg-1.1 r2 = 0.84 0.00 0.20 0.40 0.60 0.80 1.00 1.20 02004006008001,0001,200 Total Ca, Mg (mmol kg-1)WEP TP-1 Mg Ca a b Figure 2-3. Relationship between (a) waterextractable P (WEP)and WECa+ WeMg over 10 extractions in feces of Holstein dairy anim als in three different physiological states and (b) proportion of fecal TP that was wate r-extractable (WEP:TP) with total Ca and Mg content in feces of Holstein animals. Circled data points represent those of the heifer group.

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36 WEP (mmol kg -1 ) 0 5 10 15 20 25 30 35 40 45 y = 6.45 ln(Ca) 8.22 r 2 = 0.74 0 5 10 15 20 25 30 35 40 45 Mg vs P Ca vs P y = 3.09 ln(Mg) 1.64 r 2 = 0.95 y = 5.451 ln(Ca) 8.89 r 2 = 0.76 WECa, WEMg (mmol kg -1 ) 050100150200WEP (mmol kg -1 ) 0 5 10 15 20 25 30 35 40 45 y = 10.4 ln(Mg) 8.97 r 2 = 0.93 y = 14.4 ln(Ca) 21.6 r 2 = 0.87WEP (mmol kg -1 )y = 4.5 ln(Mg) 1.11 r 2 = 0.89a b c Figure 2-4. Relationship between water extractable P and water extractable Ca or Mg from feces of Holstein dairy animals in three differ ent physiological states (a) heifers, (b) pregnant nonlactating (close -up) and (c) lactating.

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37 CHAPTER 3 EFFECT OF DIETARY MODIFICATION S OF CALCIUM AND MAGNESIUM ON PERFORMANCE OF LACTATING DAIRY COWS Introduction Offsite movement of P from agricultural soils receiving manure has been well documented (Eghball et al., 1996; Sims et al ., 1998). Excess P is the most common cause of eutrophication of freshwater lakes, reserv oirs, streams, and headwaters of estuarine systems (Correll, 1998). Applicat ion of manure to soils to sa tisfy P crop requirements is an approach that can minimize P buildup in soils around confined animal feeding operations; however, this approach restricts ma nure application areas or increases costs due to transportation (Dou et al., 2003). A common and successful approach to re duce P concentrations in dairy cow manure has been to reduce the concentration of dietary P (Cerosaletti et al., 2004; Valk et al., 2000; Wu et al., 2000), which reduces the most soluble P forms in manure. From a practical standpoint and base d upon both recent research (Wu et al., 2000) and the NRC (2001) recommendations, P should be fed to la ctating dairy cows at approximately 3.7 g of P kg-1 of dietary DM, a rate at which no detrimental effects on production or reproductive performance due to low dietary P have been reported (Wu and Satter, 2000). Other dietary strategies should be examined in order to reduc e the extent of P loss from manure. Dietary Ca and Mg can interact w ith P to reduce availa bility of P in the gastrointestinal tract, mainly by the forma tion of insoluble Ca and Mg phosphates which are therefore unavailable to the animal (McDowell, 2003). Ho wever, the effect of the dietary source of Ca and the amount of Ca and Mg consumed on P balance and animal performance of lactating dairy cows has not been evaluated.

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38 This experiment tested the hypotheses that changing Ca source and both Ca and Mg concentrations in diet will not: (i) have detrimental effects on animal performance parameters, (ii) affect digestib ility of other nutrients and (i ii) significantly alter P balance in cows. The specific objectives were to eval uate effects of contro lling the concentration of Ca and Mg in the diet with two differe nt Ca sources on: (i ) animal performance parameters, (ii) nutrient digestibility, and (i ii) overall P intake and output of lactating dairy cows. Materials and Methods Cows, Diets, and Facilities The experiment was conducted at the Univ ersity of Florida Dairy Research Unit (Hague) from September to November of 2005. Experimental multiparous cows (# = 24) 575 60 kg of body weight; (128 21 days in milk) were housed in a sand-bedded freestall barn with grooved conc rete floors, and fans and sp rinklers that operated when the temperature exceeded 25C. Animals had continuous access to water, were exposed to continuous lighting during night hours, and were milk ed three times daily at 0100, 0900, and 1700 h. Prior to the start of the study, co ws were trained to use electronic feed gates (Calan gates; American Calan Inc., Northwood, NH) in order to measure dry matter intake (DMI). Cows were weighed the firs t and last 2 d of eac h period at 0500 and 1700 h. Animals were fed a total mixed ration (TMR) twice daily at 0600 and 1400 h, in ad libitum amounts (5-10% orts) silage was measured for DM weekly (Koster, Koster Crop Tester, Inc.) in order to maintain the form ulated ratio of forage to concentrate. Eight dietary treatments were evaluate d in a 2x2x2 factorial design involving 2 dietary sources of Ca (CaCO3 vs. CaCl2), 2 dietary concentrations of Ca (6.0 -LoCavs. 10.0 -HiCa-g kg-1 DM basis), and 2 dietar y concentrations of Mg (2.0 -LoMgvs. 3.5 g -

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39 HiMgkg-1 DM basis). Diets were formulated to contain the same P concentration (3.7 g kg-1 DM basis). The lower dietary Ca concentr ation was selected based on NRC (2001) recommendations for lactating dairy cows. Th e increased dietary Ca concentration was chosen to limit intake of CaCl2 below 215 g d-1, therefore minimizing possible negative effects on DMI. The low dietary Mg c oncentration required no inorganic Mg supplementation to meet the animals Mg requirement (NRC, 2001). The greater dietary Mg concentration is used in Florida to compensate for reduced Mg absorption when increased dietary concentrations of K are fed during heat stress condi tions. List of dietary ingredients and dietary chemical analysis is provided in Table 3-1 and Table 3-2, respectively. Calcium availability in the GIT was modi fied by: (a) using 2 Ca sources (CaCO3 or CaCl2) with different Ca availability coefficien t and (b) using two calcium concentrations in the diet. Inorganic Ca supplements se lected were the industry standard (CaCO3) and an anionic salt (CaCl2). Calcium chloride has been used in diet formulation to obtain a negative anion-cation balance, specific for pregnant nonlac tating dairy cows close to parturition. Nutritionally, the main differen ce between these two Ca sources is their extent and site of availabili ty. The NRC (2001) assigns an availability coefficient of 0.95 to CaCl2 and 0.75 to CaCO3. The digestive site of solubility was evaluated with an invitro study using the method developed by Moore and Dunham (1971). Results demonstrated that both sources are dissolved to 100%, but the site of availability is different. In the rumen all of CaCl2 dissolved whereas 68% of CaCO3 was soluble in the rumen incubation, the remaining Ca from CaCO3 was soluble in post-rumen digestion (upon addition of HCl and pepsin).

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40 Treatments (diets) were assigned randomly to cows in three 21-d periods. During the experiment each cow received a trea tment only once and no treatment followed another treatment from the previous period mo re than once. During the first 11 d of each period cows were adjusted to a new diet and the last 10 d were used for data collection. Sample Collection and Analysis The daily DMI was measured for individua l cows by recording the amount of TMR offered and refused. A concentrate mix contai ning the formulated mineral treatments was prepared in 0.9 tonne amounts as needed a nd stored in 1.7 tonne capacity metal bins. Concentrate, silage, and hay were mixed as TMR for each feeding. Representative samples of concentrate mixes, corn silage, and alfalfa hay were collected weekly and composited by experimental period for chemical analysis. Corn silage and alfalfa hay samples were dried at 55C in a forced-air oven and ground to pass a 1-mm screen of a Wiley mill (A.H. Thomas, Philadelphia, PA) prior to compositing. Composited and dried feed samples were analyzed for DM (105C fo r 8 h), neutral detergent fiber (NDF) using heat-stable -amylase (Goering and Van Soest, 1970; Van Soest et al., 1991), acid detergent fiber (ADF) (AOAC, 1990), a nd total nitrogen (N) (Elementar Analysensysteme, Hanau, Germany); crude protein (CP) was calculated by multiplying N x 6.25. Crude protein was calculated by mu ltiplying N x 6.25. In addition, composites were sent to a DHIA Forage Testing Laboratory (Dairy One, Ithaca, NY) where samples were analyzed by wet chemistry for Ca, P, M g, K, Na, Zn, Cu, Mn, and Fe by the ignition method (Andersen, 1976); Cl was determined by titration with AgNO3 using Brinkman Metrohm 716 Titrino Titration Unit with silver electrode (Metrohm Ltd., C-H-9101 Herisau, Switzerland) and S by oxidation (Lec o Model SC-432, Leco Instruments, Inc).

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41 Milk yields were measured for all milk ings during the collection period and milk composition (protein, fat, and somatic cells) was measured on two consecutive milkings during the last 3 d of each peri od (n=6). Somatic cell scores were generated as described by Norman et al. (2000) for statistical anal ysis of SCC. Samples were analyzed by Southeast Dairy Labs (McDonough, GA) by infrared technologies (Bentley 2000, Bentley Instruments, Chaska, MN). Average daily milk production was used to calculate average daily water intake using the equation developed by Murphy et al. (1983). The equation is as follows: Free water intake (kg d-1) = 15.99 + (1.58 x DMI, kg d-1) + (0.9 x milk, kg d-1) + (0.5 x Na intake, g d-1) + (1.20 x min temp C). Temperatur e values used for the calculation were taken from the Florida Agricultural Weather Network (FAWN) weather station in Alachua, FL. Water samples were collected from wa ter troughs every other day during the collection phase of each period and compos ited within period. Urine samples were collected from each cow at 0500 and 1300 h durin g the last 3 d of each collection period and composited within cow and period. Determination of urine pH was done as samples were collected (Horiba twin pH meter B213, Spectrum Technologies, Inc, Plainfield, IL). Water and urine samples were frozen until analysis then filtered using Whatman 42 filter paper. Calcium and Mg were measured by atomic absorption (220 FS, Varian Inc.). Phosphorus was determined on a UV-visibl e recording spectrophotometer at 880 nm wave-length via the molybdate-blue colori metric method (Murphy and Riley, 1962) (U.S. EPA, 1993; method 365.1). Colorimetric determ ination of creatinine in urine was done

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42 based on the procedure described by Vagnoni et al. (1997). Urine volume was calculated based on concentration of crea tinine in urine concentration using the equation developed by Valadares et al. (1999). Th e equation is as follows: Urine output (kg d-1) = animal weight in kg x (29.0 = creatinine concentration in urine as mg L-1) x (0.9595, L kg-1) Fecal output was calculated using the mark er ratio technique with chromic oxide (Cr2O3) as an inert dietary marker. Cows we re dosed orally via balling gun (Ideal Instruments, Inc.) with gelatin capsu les (Torpac Inc.) containing 10 g of Cr2O3 at 0500 and 1700 h from d 11 to 20 of each experi mental period. At the time of dosing Cr2O3, fecal grab samples were collected from each cow twice daily during the last 10 d of each period and composited within cow for each ex perimental period. Samples were dried at 55C for 72 h and ground through a 2-mm sc reen of a Wiley mill (A.H. Thomas, Philadelphia, PA). Fecal samples were analyzed in triplicates for total P, Ca and Mg by the ignition method (Anderse n, 1976). Total Ca and Mg were measured by atomic absorption spectroscopy; P was determined on a UV-visible record ing spectrophotometer at 880 nm wave-length via the molybdate-blu e colorimetric method (Murphy and Riley, 1962) (U.S. EPA, 1993; method 365.1). Feces were analyzed for Cr by atomic spectrophotometry (Williams et al., 1962). Appare nt digestibility of Ca, Mg, P, CP, NDF, ADF and DM were calculated usi ng the marker ratio technique. Intake of Ca, Mg, and P from water wa s calculated based on water intake and mineral concentration measured in water samples. Urine output of Ca, Mg, and P was calculated based on urine volume and mineral co ncentration measured in urine samples.

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43 Blood was collected (~10 ml) by coccygeal ve nipuncture at 1800 h into heparinized tubes (Vacutainer Company) from indivi dual cows on day 20 of each period. Blood was centrifuged immediately after collection at 1,000 x g and 4C for 15 min to separate plasma which was stored at 5C until analyzed. Concentrati ons of plasma glucose and urea nitrogen were determined with an automa ted colorimetric procedure, which utilized an autoanalyzer (AutoAnalyzer II, Bran +Luebbe, Buffalo Grove, IL). The glucose analysis is based upon the procedure desc ribed by Gochman and Schmitz (1972). The procedure for determination of urea nitrogen (Industrial Method US-339-01, Bran+Luebbe, Buffalo Grove, IL) is based on that of Coulombe and Favreau (1963). Overall P, Ca and Mg balance was calculate d by adding together intake in feed and water, then subtracting excretion in feces, urin e and secretion in milk. Milk P, Ca and Mg concentrations used to calculate minera l output in milk were: 0.93, 1.19, and 0.013 g kg-1 of milk, respectively (Na tional Dairy Council, 1993). Statistical Analysis Treatments were arranged in a 2x2x2 factor ial design using an incomplete, partially balanced Latin square. Data was analy zed using the MIXED procedure of SAS. Orthogonal single degree of freed om contrasts were used to detect main effect of Ca source, Ca concentration, and Mg concentra tion as well as 2and 3-way interactions. Differences discussed in the text were significant at P 0.05 unless otherwise indicated. The statistical model used to analyze the data was: Yijk = + i + bj + ck + eijk Where: Yikj = observed response, = overall mean, i = fixed effect of treatment i, bj = random effect of cow j,

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44 ck = fixed effect of period k, and eijk = residual error. Before completion of the third period, cow 4499 consuming diet 3, was removed from the experiment for health reasons. Results and Discussion Diet Composition and Intake Diets were formulated to contain the sa me concentration of nutrients with the exception of Ca and Mg (Table 3-2). Ch emical composition of the diets met the minimum nutrient recommendations for cows in this study based on NRC Nutrient Requirements of Dairy Cattle Software (2001). Dietary Ca and Mg concentrations varied with respect to the targeted values. Desire d LoCa concentration was 6.0 and averaged 6.4 g Ca kg-1 diet DM and targeted HiCa concen tration was 10 and ranged from 8.6 to 10.3 g Ca kg-1 diet DM; whereas LoMg diets were form ulated to contain 2.0 but averaged 2.5 despite no addition of inorgani c Mg source. The HiMg was ta rgeted to contain 3.5 g Mg kg-1 diet DM and the actual concentration ranged from 3.7 to 4.3 g Mg kg-1 diet DM. Measured dietary concentration of Ca and Mg reflect differences with respect to the formulated rations. These differences were prod uct of the combined variability present in natural feedstuffs and concentr ation of these two minerals in the different mineral mixes. These differences did not affect the main hypothesis or the objective of the study. Use of CaCl2 as a dietary anionic salt in the la te prepartum period of dairy cows to induce metabolic acidosis and promote Ca m obilization from bone is a well-documented practice (Goff and Horst, 1993; Goff et al., 2004; Pehrson et al., 1998; Tucker et al., 1991). Metabolic acidosis produced by feeding CaCl2 to dairy cows can be reflected in a reduction in urinary pH (Goff et al., 2004). Feeding CaCl2 reduced urine pH only in the

PAGE 45

45 HiCa diets. There was a dietary Ca source by Ca c oncentration interaction ( P <0.0001); When cows were fed LoCa diets urine pH wa s the same for both Ca sources. However, when cows consumed the HiCa diet c ontaining CaCl2 urine pH decreased compared with that of animals fed the high CaCO3 diet (8.1 vs. 7.0). Ther efore cows consuming CaCl2 at 9.9 g kg-1 of DM were more likely ex periencing metabolic acidosis. Animals consuming CaCl2 as the inorganic Ca supplement tended ( P = 0.0816) to experience reduced DMI when expressed quantitatively (22.0 vs. 21.2 kg d-1) but not when expressed as a proportion of BW (3.69 vs. 3.58%) compared to those fed CaCO3. Feeding CaCl2 increased the dietary concentration of Cl from 0.4 to a mean of 1.0% (DM basis). Using empirical mode ling techniques to evaluate cow responses to increasing dietary Cl concentration from several studi es, Sanchez et al. (1994) reported that DMI decreased dramatically (about 2.5 kg d-1) during the summer season but decreased very little during the winter season (about 0.5 kg d-1) when dietary Cl increased from 0.4 to 1%. The current study was conducted in the fa ll without heat stress effects on the cows; thus cow response was similar to that reported for the winter season. Glucose is the major carbohydrate source for mammalian cells to produce energy. Because of its importance, ruminants finely regulate stable plasma glucose concentrations; nevertheless, plasma glucos e can reflect the animals energy status. Average plasma glucose concentrations of animals in this ex periment (63.8 mg dL-1) were in the expected range (Dukes and Swenson, 1984), and well above those reported for hypoglycemic dairy cows (<20 mg dL-1) (Hayirli et al., 2002). Coinciding with lower DMI, cows fed CaCl2 tended ( P = 0.061) to have lower concen trations of plasma glucose (64.7 vs. 63.0 mg dL-1). Effects of feeding CaCl2 at concentrations us ed in HiCa diets in

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46 this experiment should be evaluated over l onger periods of time to assess milk production and reproductive performance. Blood urea N (BUN) can be used as an i ndicator of rumen N (protein) utilization. Concentrations of BUN were pos itively associated with intakes of ruminally degradable and undegradable protein and negatively associ ated with intake of net energy (DePeters and Ferguson, 1992). No differences among tr eatments were detected for BUN using the orthogonal single degree of freedom contrasts. The overall mean va lue (10.2 mg/dl) is within the expected range and consistent with values others have reported for lactating dairy cows in confinement (Colmenero a nd Broderick, 2006; Gressley and Armentano, 2007) and grazing with TMR supplementation (Gehman et al., 2006). Body weight and body weight change were affected by the interaction between Ca and Mg dietary concentration. Cows fed diets of greater concentrati on of Mg gained less BW when dietary concentrations of Ca were at 0.64% but not when they were at 0.95% (Ca concentration by Mg concentration interaction, P = 0.003; Table 3-3; Figure 3-1). As a result, mean BW of cows fed this combin ation of high Mg and low Ca was lighter as well ( P = 0.01; Table 3-3; Figure 3-1). Cows fed LoCa-LoMg diets had greater BW (599 vs. 588 kg) and greater BW gain (3.77 vs. 3.64 kg 21d-1) than those receiving LoCaHiMg diets whereas dietary concentration of Mg did not influence BW (593 vs. 597 kg) or BW gain (3.57 vs. 3.58 kg per 21 d) when dietary concentration of Ca was high (Mg concentration by Ca con centration interaction, P < 0.01; Table 3-3; Figure 3-1). Therefore better gains in BW due to treatment led to heavier cows. Milk Production Parameters Cows fed the LoCa diets produced more m ilk when fed the LoMg diets compared with those fed the HiMg diets (36.8 vs. 34.1 kg d-1) whereas milk yield was unchanged

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47 by Mg diets when the diets contained a hi gh concentration of Ca (34.1 vs. 33.6 kg d-1; dietary Ca concentration by dietary Mg concen tration interaction Ta ble 3-3; Figure 3-1; P =0.0136). Production of 4% fatcorrected milk tended ( P = 0.0665) to follow this same pattern (Table 3-3). The observed trend in milk yield was significantly correlated (r2=0.27) with DMI, a factor closely associat ed with milk production. Efficiency of production of uncorrected or fat-corrected milk (milk production/feed intake) was not affected by dietary treatments, averag ing 1.62 and 1.39, respectively (Table 3-3). Concentration of milk fat tended to be lower for cows fed CaCl2 (3.00 vs. 3.15%, Table 3-3; P = 0.0621). As a result, daily production of milk fat tended to be lower (0.57 vs. 0.60 kg d-1, Table 3-3; P = 0.0528) as well as production of 4% fat-corrected milk (64.2 vs. 66.7 kg d-1, Table 3-3; P = 0.0798) by cows fed CaCl2 compared to those fed CaCO3. According to Sanchez et al. (1994), incr easing Cl in the diet from 0.4 to 1.0% decreased production of 4% fat-corrected milk from about 20.8 to 19.7 kg d-1 in the summer but did not affect 4% fat-corrected milk production significantly in the winter season until Cl concentration ex ceeded 1% of dietary DM. As with milk fat concentration, milk protein concentration was lowered when CaCl2 was fed rather than CaCO3 (2.97 vs. 3.03%, Table 3-3; P = 0.0127). In addition, concentration of milk protein was re duced from 3.03 to 2.96% as the dietary concentration of Ca increased from 0.64 to 0.95% (Table 3-3; P = 0.0057). This resulted in lower production of milk protein by cows fed the greater Ca diets (1.01 vs. 1.07 kg d-1, Table 3-3; P = 0.0026). Nutrient Digestibility Apparent digestibility values of DM, protein, ADF and NDF measured in this experiment are in the expected range for diet s fed to lactating dair y cows (Ruiz et al.,

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48 1995; Staples et al., 1997). Apparent DM a nd NDF digestibilities were not affected by main dietary factors of Ca source, Ca con centration, and Mg concentration. Apparent digestibility of dietary CP increased from 64.2% to 65.3% when dietary Mg was increased from 0.25 to 0.39% (Table 3-4; P = 0.046). Increasing Mg concentration in the diets containing CaCO3 increased ADF digestibility (42.5 vs. 44.8%); however, no change was detected in ADF a pparent digestibility when in creasing Mg concentration in the diet of animals fed CaCl2 (40.0 vs. 40.0%; Ca source by Mg concentration interaction; Table 3-4; P = 0.032). These results confirm our hypothesis that changes in Ca and Mg concentrations or Ca source in the diet as used in this experiment would not have a major impact on apparent dige stibility of other dietary nutrients. Apparent P digestibility ranged from 57.2 to 63.4% and was not affected by any of the main dietary treatments (dietary Ca sour ce and dietary concentrations of Ca and Mg) as expected. Increased Mg con centration in diets used in th is experiment are comparable to those often used to feed commercial la ctating dairy cows during summer months to prevent potential negative eff ects of increased dietary K c oncentration on Mg absorption. These dietary Mg concentrations have not been reported to affect P nutrition (Jittakhot et al., 2004). Dietary concentratio ns of Ca can negatively aff ect P absorption; however, the ratio of Ca:P is a key point with respect to absorption of both elements by the animal (McDowell, 2003). In an experiment using a Ca :P ratio similar to the highest used in our experiment (2.32 vs. 2.78), Weiss (2004) re ported no signs of P deficiency when evaluating Mg digestibility. Va lues of apparent P diges tibility obtained in our study (overall mean of 59.6% ) are similar to those reported by Martz et al (1999) in a trial with nonlactating Holstein cows and with those reported by Va lk et al. (2002) in their

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49 second year of study (59.4%), yet greater than they reported for their first year of study (43.5%) in which they used slightly different dietary ingredients and P concentrations. Their results are also consistent with those of Wu et al. (2000) w ho reported apparent P digestibility values ranging between 45 and 50% when animals were fed P close to their P requirements. Main dietary factors of Ca source, Ca concentration, and Mg concentration and their interactions did not in fluence apparent digestibilit y of Ca and Mg. Apparent digestibility values of both Ca and Mg can range widely depending on dietary concentration of energy, water, fatty acids, P, Ca, Mg,, as well as ruminal pH, animal condition and age. Vitamin D status is par ticularly important for Ca as is dietary concentration of Ca and K for Mg (Rei nhardt et al., 1988). Apparent Ca and Mg digestibility values in our experiment are sim ilar to what other researchers have reported. Calcium values were similar to those reported for lactating cows fed an alfalfa and corn silage-based diet (Martz et al., 1989); whereas Mg values were comparable with those compiled from eight experiments involving 39 dietary treatments and 162 lactating Holstein cows (Weiss, 2004). Changes in dietary Ca concentration did not affect apparent Mg digestibility, consistent with results reported by Weiss (2004 ) in which similar c oncentrations of both Ca and Mg in diets of lactating dairy cows we re used. This contrasts with the idea that Ca and Mg are antagonistic in the GIT because they compete for absorption sites in the small intestine (Alcock and Macintyre, 1962). However, it has been documented that the reticulum-rumen is the main absorption site for Mg (Tomas and Potte r, 1976) whereas Ca is absorbed mainly by the sma ll intestine (Horst et al., 1994)

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50 Balance of P, Ca and Mg Daily intake of P, Ca, and Mg was quan tified taking into account that supplied by the feed and water. No differences among di etary treatments were detected for daily DMI, water intake, fecal output and urine output (Tables 3-6 and 3-7). Feed ingredients provided all of the P and most of the Ca a nd Mg intake, with water supplying a minimal proportion. Because there was no differen ce in DMI among treatments and the proportion of P, Ca, and Mg provided by wate r was both low with re spect to total and similar among treatments, concentrations of th ese minerals in the diet were the dominant factors dictating P, Ca and Mg intake. Intake (g d-1) of P, Ca and Mg reported in this study are consistent with othe r published research where dairy cows were fed similar concentrations of those nut rients (Knowlton and Herbein, 2002; Knowlton et al., 2001; Weiss, 2004; Weiss and Wyatt, 2004). Total P intake was affected by source and c oncentration of Ca in the diet. A greater P intake was detected for animals fed CaCO3 vs. CaCl2 (83.1 vs. 79.1 g d-1; P = 0.011) and for cows fed the low vs. high Ca diets (83.5 vs. 78.7 g d-1; P = 0.004; Tables 3-6 and 3-7), an effect mainly driven by the negative effect of Hi CaCl2 diets on DMI as discussed previously. The 5 g d-1 increase in P intake by cows fed the low Ca diets was accompanied by a 5 g d-1 increase in fecal output of P by this same treatment (34.5 vs. 30.5 g d-1; P = 0.005). As a result, P balance was unchanged by dietary concentration of Ca. Increased dietary P above animal requir ements resulted in hi gher fecal P excreted, consistent with the idea that feeding P cl oser to animal requirements increases its digestibility (retention) and th erefore reduces its excretion (Dou et al., 2002). Phosphorus secreted in milk represented nearly as much P as that excreted in feces; however, feces

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51 were the main excretion route for P in ag reement with what Morse et al. (1992) had previously reported. Main dietary treatments and their interacti ons did not affect overall P balance. On average, cows excreted 32.5 g of P d-1, a value lower than the 47 g d-1 reported by Weiss and Wyatt (2004) and almost half of what Bo rucki Castro et al. (2004) reported (62 g d-1) when feeding similar dietary P concentrati ons in diets with different DCAD values. Furthermore, we compared P excretion data from our experiment with the equations developed by Weiss and Wyatt (2004). Equation A: manure P (g d-1) = -2.5 + 0.64 x P intake (g d-1); Equation B: manure P (g d-1) = 7.5 + (0.78 x P Intake (g d-1)) (0.702 x milk yield (kg d-1)). Correlation coefficients (r2) of 0.28 and 0.24 were obtained for equations A and B, respectively, between our experimental da ta and the predicted values using these equations. Differences in appa rent P digestibility between our data and that generating the equations may account for the low correlation coefficients. Calcium source had no effect on Ca intake. Greater Ca concentrations were present in the CaCl2 diets; however, cows fed the CaCl2 diets had a tendency to eat less DM. Increased concentration compensated for the reduced DMI, thus there was no difference in total Ca intake when comparing two Ca s ources. Urinary concentration of Ca (0.19 vs. 0.01 g kg-1) and excretion of Ca in the urine (7.0 vs. 1.0 g d-1) was greater for cows fed the diets containing the greater concentration of CaCl2 compared with cows fed diets with the lower CaCl2 concentration; whereas these measures were not different when cows were fed the low or high concentrations of CaCO3, illustrating the greater solubility of Ca

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52 in the CaCl2 form in the digestive tract compared with that of CaCO3 (Ca source by Ca concentration interaction, P < 0.0001). Once absorbed, excess Ca can be filtered by the kidney and excreted via urine as a means to maintain Ca homeostasis; however, it is possible that a portion of that Ca (urinary excretion) can come from bone mineralization, a documented effect when anionic salts (like CaCl2) are fed to lactating dairy cows (Block, 1984; Oetzel et al., 1991). Nevertheless, the balanc e of Ca was not different between cows fed the 2 sources of Ca because urinary Ca was such a small part of the total Ca excretion. As expected, increasing Ca concentration in the diet increased Ca intake (200 vs. 139 g d-1; P < 0.0001) and fecal output (142 vs. 95 g d-1; P < 0.0001). As a result cows fed diets of 0.95% Ca tended to excrete 12 g d-1 more Ca than cows fed diets of 0.64% Ca (57 vs. 45 g d-1; P = 0.057; Tables 3-8 and 3-9). Calciu m homeostasis principles indicate that animals consuming Ca below its Ca requirement will increase the proportion of dietary Ca absorbed. However, diets containi ng more Ca than needed result in a reduced proportion of dietary Ca absorbed (Horst, 1986; Reinhardt et al ., 1988). Based on NRC (2001) recommendations, Ca concen trations in the high Ca diet s were in excess of cows requirements. Cows consuming more Mg were in a more positive Ca balance (16.6 vs. 3.6 g d-1; P = 0.034; Tables 3-8 and 3-9) but this 13-g difference simply may have been due to the tendency of a greater intake of Ca by cows fe d the diets of greater Mg concentration (173 vs. 166 g d-1; P = 0.103). Consistent with previous reports (K nowlton et al., 2001), feces was the main excretion route for Mg. Urinar y excretion of Mg was greater proportionally, than urinary

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53 excretion of both P and Ca, results consistent with other reports on lactating (Knowlton et al., 2001) and nonlactating (Jittakhot et al ., 2004) dairy cows, possibly because Mg absorbed in excess of Mg requirements is mainly excreted via urine (McDowell, 2003; Wang and Beede, 1992). Excess intake of P is recycled via saliva and increased in plasma. Excess Ca absorption triggers a se t of mechanisms to both excrete excess Ca (either via urine or feces) and to decreas e its absorption from the GIT (Horst, 1986). Consuming more Mg resulted in gr eater Mg retention (22 vs. 9 g d-1; P < 0.0001); however, this increase in Mg retention wa s greater when cows also were consuming CaCO3 (27 vs. 9 g d-1) rather than CaCl2 (17 vs. 10 g d-1; Ca source by Mg concentration interaction, P = 0.006; Tables 3-10 and 3-11, Figure 3-1). This was likely due to the greater concentration of dietary Mg in the CaCO3 diets (4.15 vs. 2.4 mg kg-1) vs. the Mg in the CaCl2 diets (3.75 vs. 2.65 mg kg-1; Ca source by Mg concentration interaction, P < 0.0001). This led to a moderately greater in take of Mg by cows fed the high Mg diets containing CaCO3 (91 vs. 54 g d-1) compared with cows fed the high Mg diets containing CaCl2 (77 vs. 56 g d-1; Ca source by Mg con centration interaction, P < 0.0001). Conclusions Increasing the Cl concentration of the diet from 4.0 to 10.0 g kg-1 (DM basis) using CaCl2 reduced urine pH from 8.1 to 7.0, indica ting that lactating cows were in a metabolic acidotic state. Cows fed diets in which supplemental CaCO3 replaced CaCl2 tended to produce more fat-corrected milk, mo re milk fat, and milk with a greater concentration of fat, as well as to produce m ilk with a greater concentration of protein. The Ca in CaCl2 appeared to be more avai lable than the Ca in CaCO3 based upon the increased concentration of Ca in the urine of cows fed CaCl2. Milk production was greatest for cows fed diets of 6.4 g of Ca kg-1 DM and 2.5 g of Mg kg-1 DM. Apparent

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54 digestibility of DM, CP, NDF, and ADF were not affected by Ca source, Ca concentration, or Mg concentra tion. Retention of P was not a ffected by diet. Cows that consumed more Ca and Mg than required excr eted and retained more of these minerals daily. Addition of CaCl2 to rations of lactating dairy cows warrants long term studies because of the tendency to decrease DMI and associated implications observed in this study.

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55 36.8 33.6 34.134.1 32.0 33.0 34.0 35.0 36.0 37.0 38.0 Low CaHigh Ca Dietary Ca ConcentrationMilk Yield (kg d-1) Low Mg High Mg 599 593 588 597 580 585 590 595 600 Low CaHigh Ca Dietary Ca ConcentrationBody Weight (kg) 3.77 3.57 3.64 3.58 3.5 3.5 3.6 3.6 3.7 3.7 3.8 3.8 Low CaHigh Ca Dietary Ca ConcentrationBody Weight Change (kg 21d-1) 36.8 33.6 34.134.1 32.0 33.0 34.0 35.0 36.0 37.0 38.0 Low CaHigh Ca Dietary Ca ConcentrationMilk Yield (kg d-1) Low Mg High Mg 599 593 588 597 580 585 590 595 600 Low CaHigh Ca Dietary Ca ConcentrationBody Weight (kg) 3.77 3.57 3.64 3.58 3.5 3.5 3.6 3.6 3.7 3.7 3.8 3.8 Low CaHigh Ca Dietary Ca ConcentrationBody Weight Change (kg 21d-1) Figure 3-1. Interaction effect of Ca by Mg dietary concentrations in a) milk yield, b) body weight, and c) body weight change.

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56 8.18.1 8.2 7.00 2 4 6 8 10 12CaCO3CaCl2 Dietary Ca ConcentrationUrine pH Low Ca High Ca 42.5 40 44.8 4037 38 39 40 41 42 43 44 45 46CaCO3CaCl2 Dietary Ca ConcentrationADF Dig. (g 100 g-1 of DM) Low Mg High Mg 12.7 31.6 14.1 21.7 0 5 10 15 20 25 30 35Low MgHigh Mg Dietary Mg ConcentrationMg Retained (g d-1) CaCO3 CaCl2 CaCO3 CaCl2 CaCO3 CaCl2 Dietary Ca Concentration CaCO3 CaCl2 8.18.1 8.2 7.00 2 4 6 8 10 12CaCO3CaCl2 Dietary Ca ConcentrationUrine pH Low Ca High Ca 42.5 40 44.8 4037 38 39 40 41 42 43 44 45 46CaCO3CaCl2 Dietary Ca ConcentrationADF Dig. (g 100 g-1 of DM) Low Mg High Mg 12.7 31.6 14.1 21.7 0 5 10 15 20 25 30 35Low MgHigh Mg Dietary Mg ConcentrationMg Retained (g d-1) CaCO3 CaCl2 CaCO3 CaCl2 CaCO3 CaCl2 Dietary Ca Concentration CaCO3 CaCl2 Figure 3-2. Interaction effect of a) Ca source by Ca concentration on urine pH and Ca source by Mg concentration on b) acid detergent fiber (ADF) digestibility, and c) Mg balance (Mg retained).

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57 Table 3-1. Ingredients and thei r concentration in dr y matter basis used in formulation of diets fed to lactating dairy cows. Ingredient g kg-1 DM Corn silage 400 Alfalfa hay 120 Ground corn 220 Whole cottonseed 100 Extruded soybean meal 60 Soybean meal 60 Minerals and vitamins premix 40 Vitamin and vitamin premixes were adde d in the same proportion to all diets.

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58Table 3-2. Chemical composition of diets fed to lactating dairy cows. Source of Ca, concentration of Ca, and concentration of Mg in diets CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 Chemical Composition CP, g kg-1 DM 161 160 161 157 158 158 159 161 ADF, g kg-1 DM 203 202 202 202 204 200 202 199 NDF, g kg-1 DM 354 347 355 344 352 355 354 352 Non-fiber carbo-hydrates, g kg-1 DM 394 398 384 390 393 381 385 365 Ash, g kg-1 DM 57 60 66 74 63 72 68 87 P, g kg-1 DM 3.7 3.8 3.9 3.8 3.8 3.7 3.8 3.7 Ca, g kg-1 DM 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Mg, g kg-1 DM 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 K, g kg-1 DM 12.0 12.0 13.0 13.0 13.0 13.0 12.0 13.0 Na, g kg-1 DM 5.0 4.0 4.0 5.0 4.0 5.0 4.0 5.0 S, g kg-1 DM 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Cl, g kg-1 DM 4.0 4.0 4.0 4.0 7.0 14.0 8.0 12.0 Fe, mg kg-1 DM 160 153 179 186 183 157 168 159 Zn, mg kg-1 DM 137 119 183 154 279 112 144 100 Cu, mg kg-1 DM 41.2 32.9 52.8 51.4 66.4 35.7 40.0 28.1 Mn, mg kg-1 DM 91.7 79.6 120 119.0 244.0 82.9 107.0 70.1

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59Table 3-2. Least squared means and standard error of the mean for animal production pa rameters of lactati ng dairy cows receivi ng two Ca sources, two Ca concentrations, and two Mg concentrations. Source of Ca, concentration of Ca, a nd concentration of Mg in diets CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 SEM DMI, kg day-1 22.5 21.8 21.7 21.9 22.6 20.4 21.0 20.7 0.64 DMI, % of BW 3.8 3.7 3.7 3.7 3.8 3.5 3.6 3.5 0.12 Body Weight, kg 603 594 588 600 596 592 588 594 4.3 BW Change, kg 21 d-1 19.8 6.6 -1.0 17.3 6.0 10.9 -9.2 16.3 5.9 BUN, mg 100ml-1 10.8 9.5 10.6 10.9 10.2 9.4 10.6 10.0 0.8 Glucose, mg 100ml-1 65.1 65.4 64.7 63.6 62.0 62.1 62.8 65.0 1.3 Urine pH 8.1 8.2 8.1 8.1 8.1 6.8 8.0 7.2 0.15 Milk yield, kg day-1 35.9 34.0 34.5 34.9 37.6 33.2 33.7 33.2 0.9 Milk Efficiency 1.6 1.6 1.6 1.6 1.7 1.6 1.6 1.6 0.5 Milk fat, % 3.12 3.163.273.053.02 3.032.89 3.08 0.11 Milk Fat, kg day-1 0.61 0.590.620.590.62 0.550.53 0.57 0.03 Fat Corrected Milk (FCM) 67.9 65.6 67.3 66.1 70.2 62.2 61.3 63.0 2.1 FCM Efficiency 1.40 1.371.421.381.42 1.381.33 1.41 0.49 Milk Protein, % 3.07 2.993.082.963.00 2.982.98 2.92 0.03 Milk Protein, kg day-1 1.09 1.051.061.041.12 0.981.00 0.97 0.03 Somatic cell count (x1000) 165 110 191 165 361 189 68 159 17.8

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60Table 3-3. P values for animal production parameters of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 DMI, kg day-1 0.0816 0.1122 0.2728 0.2318 0.6965 0.1135 0.6064 DMI, % of BW 0.1483 0.1163 0.3867 0.4143 0.8613 0.3666 0.5656 Body Weight, kg 0.2239 0.7018 0.9532 0.2574 0.8449 0.01 0.4133 BW Change, kg 21 d-1 0.2707 0.0458 0.1563 0.2251 0.9881 0.0032 0.5436 BUN, mg 100ml-1 0.4411 0.2842 0.8294 0.3194 0.9688 0.379 0.5153 Glucose, mg 100ml-1 0.0607 0.6623 0.3953 0.6744 0.1278 0.8494 0.3278 Urine pH <.0001 0.0002 <.0001 0.6658 0.6604 0.3374 0.2451 Milk yield, kg day-1 0.4704 0.0156 0.1825 0.0764 0.1904 0.0136 0.5104 Milk Efficiency 0.3374 0.8568 0.8959 0.9257 0.6298 0.4118 0.7512 Milk fat, % 0.0621 0.8932 0.2468 0.896 0.7129 0.7893 0.1708 Milk Fat, kg day-1 0.0528 0.3357 0.8632 0.2912 0.3158 0.216 0.1458 Fat Corrected Milk 0.0798 0.1179 0.6606 0.152 0.1959 0.0665 0.1665 FCM Efficiency 0.922 0.8689 0.4805 0.7903 0.5864 0.4608 0.3695 Milk Protein, % 0.0127 0.0057 0.176 0.3078 0.5411 0.436 0.9999 Milk Protein, kg day-1 0.1165 0.0026 0.3866 0.0743 0.1305 0.0518 0.4912 Somatic cell count 0.8076 0.7327 0.4744 0.200 0.280 0.0773 0.2469

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61Table 3-4. Least squared means and standard error of the mean for nutri ent digestibility of lactating dairy cows receiving two Ca sources, two Ca concentrations and two Mg concentrations. Ca Source, dietary Ca concentration, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 SEM Digestibility (%) DM 70.3 69.6 70.6 70.4 69.0 68.9 68.4 69.8 2.6 Protein 65.0 64.4 66.8 64.7 64.3 63.1 65.3 64.6 4.1 ADF 42.4 42.5 44.7 44.9 37.1 43.0 36.8 43.1 3.5 NDF 46.8 47.1 49.0 49.1 44.9 48.1 43.7 49.1 1.3 Phosphorus 57.7 63.4 58.1 58.7 57.2 61.4 59.1 61.2 1.4 Calcium 23.4 28.7 39.6 29.5 30.9 23.5 29.7 31.2 3.1 Magnesium 22.3 31.0 37.3 41.2 35.7 34.1 37.0 35.0 2.7

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62Table 3-5. P values for nutrient digestibility of la ctating dairy cows receiving two Ca sour ces, two Ca concentrations, and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 DM 0.887 0.100 0.987 0.715 0.433 0.327 0.706 Protein 0.613 0.369 0.927 0.046 0.397 0.566 0.055 ADF 0.316 0.373 0.128 0.008 0.032 0.602 0.675 NDF 0.175 0.917 0.566 0.670 0.807 0.579 0.797 Phosphorus 0.338 0.255 0.853 0.234 0.917 0.789 0.628 Calcium 0.105 0.172 0.195 0.597 0.600 0.942 0.979 Magnesium 0.427 0.250 0.308 0.596 0.585 0.779 0.781

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63Table 3-6. Least squared means and standard error of the mean for dry matter and wa ter intake, urinary a nd fecal output and ov erall P retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations. Ca Source, dietary Ca concentration, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 SEM DMI, kg d-1 22.5 21.821.721.922.6 20.421.0 20.7 0.64 Water intake, kg d-1 104 101 96.3105 98.0 103 104 96.5 3.8 Fecal output, kg d-1 6.7 6.56.36.46.9 6.36.6 6.2 0.2 Urine output, kg d-1 40.3 49.551.538.548.8 42.833.2 36.2 9.9 Phosphorus Feed, g kg-1 3.8 3.8 3.9 3.7 3.8 3.7 3.8 3.7 0.0 Water, g kg-1 ---------Feces, g kg-1 5.3 4.6 5.5 5.2 5.3 4.7 5.0 4.8 0.2 Urine, g kg-1 ---------Intake in feed, g d-1 85.0 83.0 84.0 81.0 86.0 75.0 79.0 76.0 2.1 Intake in water, g d-1 ---------Output in feces, g d-1 36.0 30.0 34.0 34.0 36.0 29.0 32.0 29.0 2.0 Output in urine, g d-1 0.1 0.1 0.1 0.0 0.1 0.1 0.0 0.0 0.0 Output in milk, g d-1 32.2 31.1 30.3 34.3 31.4 32.8 30.8 30.5 2.3 Retention, g d-1 17.2 21.9 19.2 12.9 17.1 13.0 16.0 16.3 3.4

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64Table 3-7. P values for dry matter and water intake, urinar y and fecal output and overall P balance (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concen trations, and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 DMI, kg d-1 0.082 0.112 0.273 0.232 0.697 0.114 0.606 Water intake, kg d-1 0.584 0.710 0.422 0.625 0.719 0.917 0.038 Fecal output, kg d-1 0.863 0.071 0.176 0.114 0.747 0.391 0.991 Urine output, kg d-1 0.507 0.815 0.980 0.424 0.461 0.633 0.292 Phosphorus Feed, g kg-1 0.033 0.001 0.807 0.996 0.887 0.040 0.007 Water, g kg-1 -------Feces, g kg-1 0.202 0.009 0.941 0.442 0.147 0.191 0.910 Urine, g kg-1 0.231 0.408 0.801 0.281 0.257 0.540 0.728 Intake in feed, g d-1 0.011 0.004 0.194 0.199 0.620 0.286 0.130 Intake in water, g d-1 -------Output in feces, g d-1 0.247 0.005 0.505 0.812 0.331 0.076 0.968 Output in urine, g d-1 0.469 0.907 0.358 0.186 0.948 0.676 0.939 Output in milk, g d-1 0.706 0.539 0.798 0.804 0.516 0.580 0.318 Retention, g d-1 0.364 0.577 0.830 0.612 0.369 0.473 0.134

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65Table 3-8. Least squared means and standard error of the mean for dry matter and wa ter intake, urinary a nd fecal output and ov erall Ca retention (g d-1) of lactating dairy cows receivi ng two Ca sources, two Ca concentra tions, and two Mg concentrations. Ca Source, dietary Ca concentration, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 SEM DMI, kg d-1 22.5 21.8 21.7 21.9 22.6 20.4 21.0 20.7 0.64 Water intake, kg d-1 104 101 96.3 105 98.0 103 104 96.5 3.8 Fecal output, kg d-1 6.7 6.5 6.3 6.4 6.9 6.3 6.6 6.2 0.2 Urine output, kg d-1 40.3 49.5 51.5 38.5 48.8 42.8 33.2 36.2 9.9 Calcium Feed, g kg-1 6.0 8.6 6.5 9.5 6.4 9.5 6.4 10.2 0.15 Water, g kg-1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.00 Feces, g kg-1 15.6 20.2 13.2 22.8 14.8 23.6 14.9 23.4 0.86 Urine, g kg-1 0.03 0.030.000.020.020.190.010.18 0.02 Intake in feed, g d-1 141 188 140 209 142 195 135 209 5.61 Intake in water, g d-1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.18 Output in feces, g d-1 105 132 80 146 99 148 96 143 6.36 Output in urine, g d-1 1.0 1.0 1.0 1.0 1.0 8.0 1.0 6.0 0.89 Output in milk, g d-1 41.2 39.7 38.8 43.9 40.2 42.0 39.4 39.0 2.9 Retention, g d-1 -3.9 17.8 19.8 21.4 2.2 -1.5 1.7 23.6 8.7

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66 Table 3-9. P values for dry matter and water intake, urinary and fecal output and overall Ca retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concen trations, and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 DMI, kg d-1 0.082 0.112 0.273 0.232 0.697 0.114 0.606 Water intake, kg d-1 0.584 0.710 0.422 0.625 0.719 0.917 0.038 Fecal output, kg d-1 0.863 0.071 0.176 0.114 0.747 0.391 0.991 Urine output, kg d-1 0.507 0.815 0.980 0.424 0.461 0.633 0.292 Calcium Feed, g kg-1 <.0001 <.0001 0.003 <.0001 0.170 0.006 0.607 Water, g kg-1 0.927 0.519 0.523 0.997 0.205 0.636 0.059 Feces, g kg-1 0.045 <.0001 0.236 0.907 0.938 0.054 0.044 Urine, g kg-1 <.0001 <.0001 <.0001 0.391 0.733 0.644 0.889 Intake in feed, g d-1 0.879 <.0001 0.572 0.103 0.413 0.008 0.952 Intake in water, g d-1 0.798 0.745 0.752 0.743 0.259 0.444 0.177 Output in feces, g d-1 0.200 <.0001 0.857 0.275 0.899 0.035 0.037 Output in urine, g d-1 0.001 <.0001 <.0001 0.096 0.431 0.708 0.798 Output in milk, g d-1 0.706 0.539 0.798 0.804 0.516 0.580 0.318 Retention, g d-1 0.243 0.103 0.840 0.034 0.912 0.821 0.082

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67Table 3-10. Least squared means and SEM fo r dry matter and water intake, urinary and fecal output and overall Mg retention (g d-1) of lactating dairy cows receiving two Ca sources, tw o Ca concentrations, and two Mg concentrations. Ca Source, dietary Ca concentration, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 SEM DMI, kg d-1 22.5 21.8 21.7 21.9 22.6 20.4 21.0 20.7 0.64 Water intake, kg d-1 104 101 96.3 105 98.0 103 104 96.5 3.8 Fecal output, kg d-1 6.7 6.5 6.3 6.4 6.9 6.3 6.6 6.2 0.2 Urine output, kg d-1 40.3 49.5 51.5 38.5 48.8 42.8 33.2 36.2 9.9 Magnesium Feed, g kg-1 2.3 2.5 4.4 4.0 2.6 2.7 3.7 3.8 0.04 Water, g kg-1 0.01 0.010.010.010.010.010.010.01 0.00 Feces, g kg-1 6.1 5.6 9.4 7.9 5.3 5.8 7.5 8.1 0.28 Urine, g kg-1 0.10 0.110.120.150.220.170.250.27 0.03 Intake in feed, g d-1 54.0 53.5 95.0 87.8 57.1 54.5 76.9 76.5 2.0 Intake in water, g d-1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1 Output in feces, g d-1 41 36 59 51 36 36 48 50 1.9 Output in urine, g d-1 3.0 4.0 5.0 6.0 6.0 6.0 6.0 8.0 0.8 Output in milk, g d-1 4.5 4.3 4.2 4.8 4.4 4.6 4.3 4.3 0.3 Retention, g d-1 6.7 9.9 27.1 27.1 10.1 8.9 19.3 15.4 2.6

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68 Table 3-11. P values for dry matter and water intake, urinary and fecal output and overall Mg retention (g d-1) of lactating dairy cows receiving two Ca sources, two Ca concen trations, and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 DMI, kg d-1 0.082 0.112 0.273 0.232 0.697 0.114 0.606 Water intake, kg d-1 0.584 0.710 0.422 0.625 0.719 0.917 0.038 Fecal output, kg d-1 0.863 0.071 0.176 0.114 0.747 0.391 0.991 Urine output, kg d-1 0.507 0.815 0.980 0.424 0.461 0.633 0.292 Magnesium Feed, g kg-1 <.0001 0.958 0.001 <.0001 <.0001 <.0001 0.002 Water, g kg-1 0.925 0.509 0.440 0.969 0.179 0.794 0.110 Feces, g kg-1 0.005 0.313 0.001 <.0001 0.175 0.280 0.141 Urine, g kg-1 <.0001 1.000 0.532 0.055 0.544 0.335 0.564 Intake in feed, g d-1 <.0001 0.077 0.444 <.0001 <.0001 0.515 0.178 Intake in water, g d-1 0.970 0.798 0.664 0.741 0.259 0.545 0.317 Output in feces, g d-1 0.002 0.044 0.009 <.0001 0.271 0.949 0.271 Output in urine, g d-1 0.001 0.206 0.556 0.013 0.415 0.348 0.542 Output in milk, g d-1 0.706 0.538 0.798 0.804 0.516 0.580 0.318 Retention, g d-1 0.024 0.813 0.269 <.0001 0.007 0.409 0.943

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69 CHAPTER 4 DIETARY CONTROL OF CALCIUM AND M AGNESIUM AS A MEANS OF REDUCING PHOSPHORUS SOLUBILITY IN DAIRY FECES Abstract Large dietary intakes of calcium (Ca) a nd magnesium (Mg) can reduce phosphorus (P) absorption by the formation of insoluble phosphate inside the gastro inte stinal tract; however, from an environmental perspective their interact ion with P may decrease P solubility in dairy feces and minimize losses from agricultural lands The objective of this study was to evaluate effects of dietary Ca and Mg concentrations and solubility of Ca source on P concentration and solubility in feces of lactating cows receiving diets with the same P concentration. Twenty four multiparous cows in mid lactation were fed eight different diets in three-21 d periods. Fecal samples were collected twice a day during the la st 10 d of each period, composited within cow, and dried at 45 C. Successive wa ter extractions (100:1 water:fece s ratio, shaken for 1 h) were performed for all dried fecal samples. Dietary tr eatments had no effect on fecal P concentrations. Increased Ca concentration in both diet and f eces reduced water extrac table P (WEP) from fecal samples. Increased fecal concen trations of Mg and Ca in feces mutually suppressed the dissolution of each element, sugge sting their association in a so lid phase. Fecal samples with higher Ca and Mg concentrations showed re duced WEP; possibly, Ca and Mg mutually suppressed dissolution of Ca,Mg-P forms by the common ion effect. Calcium and Mg dietary modifications effectively reduced P solubility in fecal samples of lactati ng dairy cows despite the same dietary and fecal P concentration. Introduction Intensively managed animal operations import feedstuffs to satisfy dietary requirements and maximize animal production. Manure generated from these operations is often applied to soils in excess of crop requirements for phos phorus (P), leading to buildup of soil P

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70 concentrations in the long term (Sentran and Ndayegamiye, 1995; Toth et al., 2006; Whalen and Chang, 2001), increasing the potential for offsite P movement with detrimental effects on surface water quality (Pote et al., 1996; van Es et al., 2004). Manure-impacted soils often have conditions associated with increased P stability (elevated concentrations of P and Ca and high pH), but no solid state crystalline phosphate minerals have been identified in these soils (Harris et al., 1994) or in (dairy) manure-soil incubations (Cooperband and Good, 2002). Instead, P re mains highly soluble, even after years of abandonment (Josan et al., 2005; Nair et al., 1995 ), where high concentrations of Mg and dissolved organic carbon have been proposed as inhibitors of more stable calcium-phosphate minerals (Ca-P) in manure-amended soils (Harris et al., 1994; Josan et al., 2005). Research efforts have focused on decreas ing P in animal diets and therefore P concentration in feces, the main route of P excr etion (Morse et al., 1992) Dietary modifications have successfully reduced fecal P concentratio ns up to 33% when used in commercial dairy farms (Cerosaletti et al., 2004), pa rticularly decreasing the watersoluble P fraction (Dou et al., 2002), with no detrimental effects on animal productivity (Wu and Satter, 2000). However, all of these experiments where significant reduction in manure P has been documented, initial dietary P was well above animals requirements; furtherm ore fecal P concentrations remain above 4.0 g kg-1 of DM even when cows were fe d with diets containing P at 3.1 g kg-1 of DM. There is a threshold point to which dietary and therefore manure P can be reduced. Based on National Research Council recommendations, P should be fed to lactating dairy cows at approximately 3.7 g kg-1 of diet DM (NRC, 2001). Contrasting findings on animal performance have been reported when feeding P at concentrations below 3.7 g kg-1 of diet DM. In different experiments Wu et al. (2000) documented nega tive impacts on animal performance when

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71 feeding P at 3.1 g kg-1 of diet DM, whereas Dou et al. (2002) concluded that 3.1 to 3.7 g of P kg-1 of dietary DM was adequate or near adequate for milk production. Emphasis thus far has been placed on reducing dietary and fecal P concentrations, but the solubility and long-term stability of P in manur e may be of equal or greater importance. Fecesderived components can play an impor tant role in the fate of P in soils, particularly soils with low P sorbing capacity (Josan et al., 2005). Phosphorus availability (digestibility or absorp tion) -and therefore excretionin the gastro intestinal tract (GIT) of ruminants is influenced by several factors. Di etary concentration and source of P are most influential (Chapuis-Lardy et al., 2004); however, inte stine pH, animal age, intestinal parasitism and intakes of Ca, Fe, Al Mn, K and Mg can also affect P absorption. Increased concentration of cations in the diet has been shown to decrease P availability in the GIT and thereby to increase P excretion (McDowe ll, 2003); however, effects of those cations on fecal P solubility has not been evaluated. Calcium and P nutrition are often considered to gether because they are closely related; excess of one can result in poor absorption of the other. From an animal science perspective, large intakes of Ca and Mg (as we ll as Fe and Al, usually low in dairy cow diets) are of concern because of their effect on decreasing P absorp tion by the formation of insoluble phosphate in the GIT (McDowell, 2003). From an environmenta l perspective and given the importance of reducing P losses from agricultural lands (S ims et al., 2000; USEPA, 2000), decreased P solubility in dairy manures is a desired characteristic. This study tested the hypothesis th at increased Ca availability in the GIT of lactating dairy cows will favor Ca-P formation with potential to reduce P solubility in feces. The objective was

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72 to evaluate effects of 2 dietary concentrations of Ca and Mg and solubility of dietary Ca source on P concentration and solubility in feces of lactating cows. Materials and Methods The experiment was conducted at the Univer sity of Florida Dairy Research Unit from September to November of 2005. Cows were managed according to the guidelines approved by the University of Florida Animal Care and Use Committee. Eight different diets were fed to a group of 24 multiparous cows averaging 128 (1) days in milk (DIM) and a production of 39.5 (.2) kg of milk a day. Cows were in a freestall barn with grooved concrete floors, and fans and sprinklers that operated when the temperature exceeded 25C. Animals had continuous access to water and individual pens bedded with sand and with continuous lighting during night hours, and were milked mechanically three times a day at 0500, 1300 and 2100 h. While receiving the standard farm diet, cows were trained for ten da ys to use electronic feed gates (Calan gates; American Calan Inc., Northwood, NH) to allow measurement of dry matter intake (DMI) by individual cows. Before completion of the thir d period, one of the cows was removed from the experiment for health reasons. Diets offered were formulated to cont ain the same P concentration (3.7 g P kg-1 of DM). Increased Ca availability in the GIT tract wa s achieved by: (a) using 2 Ca sources (CaCO3 or CaCl2) each having a different Ca availability coefficient and (b) using two calcium concentrations in the diet. I norganic Ca supplements selected were the industry standard (CaCO3) and an anionic salt (CaCl2). Calcium chloride is used in diet formulation to obtain desired anion-cation value, for pr egnant nonlactating dairy cows cl ose to parturition. Desired Ca concentrations were 6.0 (L oCa) and 10.0 (HiCa) g kg-1 of dietary DM. The former was selected based on NRC (2001) recommendations for lactating dairy cows; the latter, chosen to increase Ca concentration in the diet while maintaining CaCl2 at a concentration below 27.5 mg kg-1 of

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73 the diet DM and prevent possible negative effect s on DMI. Consideration was also given to the Ca:P ratio in the diet. Desi red Mg concentrations were 2.0 (LoMg) and 3.5 (HiMg) g kg-1 of diet DM. The lower Mg concentration requires no inorganic Mg supplementation while meeting animal requirements; the high Mg concentration is used in Florida to minimize negative impacts of heat stress on lactating dairy cows during the summer months. Dietary ingredients (and proporti ons) for all diets were: corn silage (40%); alfalfa hay (12%), ground corn (22%), whole cottonseed (10 %), soybean meal (6%), extruded soybean meal (6%), and vitamin and mineral premix (4%) on dr y matter basis. Treatments (diets) were assigned to cows in three 21-d periods. During the experiment each cow received a treatment only once and no treatment followed another treatmen t from the previous period more than once. During the first 11-d of each period cows were ad justed to a new diet. Fecal grab samples were collected twice a day from day 12 to day 21 to obtain a composite sample per cow per period. Individual feed ingredient were sampled on ce a week during each period and composited per period. Composited feed and fecal samples were dried at 45C in a forced-air oven and ground to pass the 2-mm screen of a Wiley mill (A.H. Thomas, Philadelphia, PA). Fecal output was calculated using the marker ratio technique with chromic oxide (Cr2O3) as an inert dietary marker. Cows were dosed with 10 g of Cr2O3 twice a day from d 11 to 20 of each experimental period. Fecal samples were dried to standardize all sa mples to the same DM content so that the same amount of moisture+DM from each fecal sample would be mixed with distilled water for extraction. Drying is also effectiv e to obtain consistency in sample treatment, preserve samples, and avoid DM variability in feces between anim als and within animals in different sampling events. Furthermore, feces (or manure) often und ergo a drying period upon application to soil.

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74 We are aware of the possible effects on P sol ubility when fecal samples are dried. Previous research has reported cont radictory findings. Ajiboye et al. ( 2000) reported an increased in water extractable P (WEP) from dairy cow manure when samples were oven-dried at 105 C whereas Chapuis-Lardy et al. (2004) showed a decrease in inorganic P soluble in water when dairy cow fecal samples were dried at 65 C. We believe that relative treatment effects would not be compromised by drying feces at low temperature, as Dou et al. (2000) found that a ~70% of P in dairy manure was water soluble and most of it was extracted in the initial steps of repeated water extractions. Ten successive water extractions were perfor med on all 71 fecal samples. A mixture of 100:1 water:feces ratio was selected to maxi mize P solubility and extraction and detect differences in WEP among treatments in the long term. After 1h of shaking, samples were centrifuged at 1000x g for 5 minutes. The supernat ants were collected and filtered through 0.45 m filter. Extractions were carried out at room temperature (~25 C). Collected supernatant solutions were analyzed for soluble reactiv e phosphorus, Ca, and Mg. Fecal samples were analyzed in triplicates for total P, Ca and Mg by the ignition method (A ndersen, 1976). Total and water-extractable Ca and Mg were measured by atomic absorption (AA) spectroscopy; P was determined on a UV-visible recording spectr ophotometer at 880 nm wave-length via the molybdate-blue colorimetric method (Murphy and Riley, 1962) (U.S EPA, 1993; method 365.1). Composited and dried feed samples were analyzed for DM (105C for 8 h), neutral detergent fiber (NDF) using heat-stable -amylase (Goering and Van Soest, 1970; Van Soest et al., 1991), acid detergent fiber (ADF) (AOAC, 1990) and total nitrogen (N); crude protein (CP) was calculated by multiplying N x 6.25(Elementar Analysensysteme, Hanau, Germany). Crude protein was calculated by multiplying N x 6.25. In addition, a composites were sent to a DHIA

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75 Forage Testing Laboratory (Dairy One, Itha ca, NY) where samples were analyzed by wet chemistry for Ca, P, Mg, K, Na, Zn, Cu, Mn, and Fe by the ignition method (Andersen, 1976); Cl was determined by titration with AgNO3 using Brinkman Metrohm 716 Titrino Titration Unit with silver electrode (Metrohm Ltd., C-H-9101 Herisau, Switzerland) and S by oxidation (Leco Model SC-432, Leco Instrument s, Inc) (Table 4-1). Fecal samples from 3 cows from each of the tw o treatments that resulted in highest and lowest WEP were selected to be analyzed by Scanning Electron Micros cope (SEM). Samples were first sieved to pass a 53 m screen. Samples were placed on a SEM carbon mount and then coated with a thin layer of carbon. The sample was analyzed with a JSM 6400 Scanning Electron Microscope unit using an accelerating voltage of 15 kV. A general dot map was first obtained from the sample; then an elemental spectral wa s obtained from areas with high P concentration. Those samples and three samples from two other diets (LoCaCO3, Hi Mg and Hi CaCO3, HiMg) were analyzed by X-ray diffraction in a dry powder mount preparation. Visual-MINTEQ Version 2.51 was used to calc ulate chemical specia tion (including solidphase equilibrium forms) under an open system for data from the first extraction (Gustafsson, 2005). In addition to P, Ca and Mg measurements for all extracts, chemical speciation data included pH and electrical conduc tivity (EC) using a standard pH and conductivity meter; Na and K by AA; and dissolved organic C dete rmined using a carbon analyzer (TOC-5050A; Shimadzu, Kyoto, Japan) (Method 5310A; Americ an Public Health Association, 1992). Statistical Analyses: Treatments were arranged in a 2x2x2 factorial design using an incomplete, partially balanced Latin square. Da ta was analyzed using the GLM procedure of SAS. Orthogonal single degree of freedom contrasts were used to detect main effect of Ca source, Ca concentration, and Mg concentra tion as well as 2and 3-way interactions.

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76 The statistical model used to analyze the data was: Yijk = + i + bj + ck + eijk Where: Yikj = observed response, = overall mean, i = fixed effect of treatment, bj = random effect of cow, ck = fixed effect of period, and eijk = residual error. Correlations and nonlinear regressions were used to describe relationships between elements in our analysis. Differences disc ussed in the text were significant at P 0.05 unless otherwise indicated. Results and Discussion Dietary and Total Fecal Concentrations of Phosphorus, Calcium, and Magnesium Several factors have been shown to affect mi neral digestibility and therefore excretion; intake amount and source, intestine pH, age of an imal, intestinal parasitism and intake level of other elements are among the most influentia l (McDowell, 2003). In this experiment fecal concentration of P was not affected by di etary concentration of Ca and Mg. Dietary concentration of Ca and Mg did not affect fecal concentration of each other. Therefore, of the measured parameters, dietary concentration was the most influential factor controlling fecal concentration of each respective mineral. No diffe rences in concentration of dietary or fecal P were detected based on the single degree of freedom contrasts used (Table 4-2 and 4-4). This is consistent with other reports when dietary and fecal P have been measured in lactating dairy cows (Morse et al., 1992) and more recently (Wu et al., 2000). Increasing dietary concentration of Mg did not affect fecal P concentration (Table 4-4). However, feeding HiCa diets, ir respectively of source, reduced concentration of P in feces (4.8 vs. 5.3 g kg-1). Contrary to our results, Steevens et al. (1971) reported that higher dietary Ca

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77 concentrations resulted in decreased P absorp tion (or increased P excreted) by lactating dairy cows. However, Borucki Castro et al. (2004) reported no differences in fecal or urine P concentrations with changes in dietary anion-cation balance for la ctating dairy cows. Deitert and Pfeffer (1993), working with milking goats over tw o lactations, found no consistent effect when doubling dietary Ca NRC recommendations on P ba lance. Additionally, Wise et al. (1963) found no detrimental effects on growth or feed effici ency of calves when dietary Ca:P ratios were between 1:1 and 7:1. Although the exact mechanism for increased fecal P w ith decreased dietary Ca is not clear, no detrimental effects on animal performance due to reduced P availability or absorption were observed with dietary Ca:P ratios used in this experiment. Dietary Ca and Mg concentrations had highe r variability than expected from ration formulation calculations (Table 4-1). Ho wever, based on NRC (2001) recommendations, measured Ca and Mg concentrations in the diet we re in a safe range to meet animal requirements without interfering with absorpti on of other nutrients. Variability of those elements in the diet was reflected by an interaction between dietar y Ca source by Ca concentration on total Mg concentration in feces ( P = 0.001), though fecal Mg concentrations closely related to dietary Mg concentration. Effects of dietary concentrations of Ca, P and Mg on Mg fecal concentrations in this experiment are consistent with those repor ted by Weiss (2004) when measuring digestibility of Mg in lactating dairy cows. Weiss found that Ca concentrations in manure reflected its dietary amounts; Ca source affected total Ca concentratio ns in manure, but this effect was driven by higher dietary Ca in diets containing CaCl2 as the inorganic Ca source. Increased fecal Ca concentrations (14.2 to 23.5 g kg-1) were greater, proportionally, than its counterparts in the diet (6.4 vs. 9.5 g kg-1), suggesting a decrease in apparent digestibility (absorption efficiency) with increased dietary Ca Calcium concentrations in the HiCa diets are

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78 above the animal requirement, such that additional intake is not absorbed as efficiently in the digestive tract. Contrary to Ca and P, apparent Mg digestibility increased with increased dietary Mg concentration, possibly because of greater ava ilability of inorganic Mg supplement (added to HiMg diets) as opposed to the Mg present in organic feedstuffs (i.e. corn silage). Effect of Dietary and Fecal Concentrations of Ca and Mg on Water Extractable P, Ca, and Mg Water extractable P data were an alyzed as concentration (mg kg-1) and standardized to TP in feces (WEP:TP). There were no differences in fecal TP among treatments, so statistical results from WEP and WEP:TP we re essentially equivalent. Ther efore, only concentration data will be discussed. Water extractable P was reduced when CaCl2 was the inorganic Ca source in the diet (as opposed to CaCO3) (1.98 vs. 2.31 g kg-1). Increasing dietary Ca con centrations, regardless of the Ca source reduced WEP (1.80 vs. 2.49 g kg-1)) (Fig. 4-1a). Increased Ca availability, not only concentration, also had an effect on reducing WEP (r2=0.37). Results from our experiment suggest that increased Ca deliver ed to the GIT, either by increa sed solubility (source effect) or increased concentration (HiCa vs LoCa) reduced WEP in feces from cows receiving those treatments, perhaps because of increased associati on of Ca with P. Increa sed Mg concentration in diet and feces did not have an effect on WEP (2.26 vs. 2.03 g kg-1) (Fig. 4-1). Cumulative water extractable Mg in feces wa s consistent with dietary and fecal Mg concentrations. Although in teractions of dietary Ca source by Ca concentration treatments and Ca concentration by Mg concentration in the di et affected fecal WEM g, these interactions reflected the variability in dietary concentrations of both Ca and Mg. Cumulative water extractable Ca concentrati on from feces was greater in samples from cows receiving HiCa diets, consistent with TC a in fecal samples. However, LoCa diets had a

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79 greater proportion extracted when data were normalized to TCa concentration in feces (WECa:TCa). Fecal samples from animals receivi ng HiMg diets had lower WECa compared to those fed LoMg diets (5.10 vs. 5.81 g kg-1), whereas diets containing CaCl2 reduced total and WEMg (5.06 vs. 5.85 g kg-1) concentration in feces. Therefore, increased fecal concentrations of Mg and Ca in feces had a synergistic effect on re ducing solubility of each other, suggesting their association in a solid phase and limited dissolution because of the common ion effect. Water extractable P, Ca, and Mg concentratio ns were also analyzed cumulatively after each individual extraction; there was no effect of dietary Ca concentration and source on cumulative WEP for the first seven extractions after the seventh ex traction, cumulative WEP from fecal samples from cows receiving HiCa diets was lower than WEP from cows fed the LoCa diets (Fig 4-2a). Increases in WEP afte r the seventh extraction were smaller for fecal samples from HiCa treatments, suggesting that rema ining P in the sample has increased stability; chemical speciation modeling indicated that Ca -P or Ca,Mg-P minerals were undersaturated based on extract solution concentr ations. Dietary Ca concentration was also the main variable affecting WECa; feces from cows receiving HiCa diets had increased WECa throughout the ten extractions. The same was true for WEMg from f eces and dietary Mg concentrations (Fig. 4-2). Relationship between Fecal WEP with WEMg and WECa Concentrations in Fecal Samples Release of P in water extractions from fecal samples was associated with that of Ca and Mg (Table 4-3). This relationship was nega tive for the first extraction where increased concentrations of Ca and Mg (mmol kg-1 of feces) reduced WEP in all treatments (r2=0.23); this trend did not change when data were analyzed by Ca source, and Ca or Mg concentration in the diet. The presence of soluble Ca and Mg salts mi ght be responsible for the high concentration of those two elements in the first extraction. Chemi cal speciation analysis of the first extraction showed that concentration of P speci es in solution was dominated by HPO4 -2 and H2PO4

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80 representing 65% or more of TP in solution; Mg-P represented ~20% and Ca-P ~10%; K and NaP combined for less than 1% of TP in solution. Th ese results are consistent with leachate data from manure amended soils using a 1:10 soil to so lution ratio (Silveira et al., 2006). Saturation indices showed solutions to be supersaturat ed with respect to wh itlockite (Ca,Mg-P) and hydroxylapatite (Ca-P), indicat ing that these minerals, if present, would not have dissolved in the first extract. Possibly, Ca and Mg together suppressed dissolution of Ca,Mg-P forms (e.g., whitlockite) by the common ion effect. Concentrations of Ca and Mg in solution af ter the first extraction were greatly reduced compared to the first extraction (Fig. 4-2); we susp ect that most of the re adily-soluble Ca and Mg salts were dissolved in the first extraction becaus e of the high solution to feces ratio used. From the second extraction onward, relationship of WE P with WECa and WEMg from fecal samples changed to a positive one, possibly due to dissolution of Ca-Mg phosphate. These results are consistent with previous reports when using hi gh water:manure ratio (K leinman et al., 2002) and when studying P release from manure-amended so ils (Hansen and Strawn, 2003; Josan et al., 2005; Silveira et al., 2006). Regression coefficients for the associati on of WEP with WECa, WEMg, or WECa+WEMg (Table 4-3) were calculated using data for extr actions 2-10. Water extractable P variability was better explained by Mg release from fecal sample s than Ca, a behavior consistent across diets with or without the inclusi on of data from the first extraction in the equation. Interactions observed between WECa and WE Mg, and the observed associated release between P, Ca, and Mg led us to use solid state assessments in fecal samples to explore reasons for the significant differences in WEP among fece s (treatments) with similar TP content.

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81 No evidence of a crystalline Ca and/or Mg phosphate mineral was detected by X-ray diffraction analysis in dried fecal samples. Howe ver, when these samples were ashed to remove organic matter, different minerals (Ca/Mg phos phate among them) were observed. sylvite (KCl) and calcite (CaCO3) were common minerals to all samples analyzed and used as internal standards. Whitlockite (Ca,Mg)3(PO4)2 was the only P-containing mi neral detected in fecal samples from animals being fed LoCaCO3, HiMg diet (Fig. 4-3.1) ; whitlockite and some hydroxyl-apatite Ca5(PO4)3(OH, Cl, F) were detected in ashed fecal samples from animals receiving diet with LoCaCO3, LoMg (Fig. 4-3.2). Feces from cows fed HiCa diets behaved differently, those from cows fed HiCaCO3, HiMg showed a dominance of hydroxyl-apatite with some withlockite (Fig. 4-3.3) whereas hydroxyl -apatite was the only P-containing mineral observed in ashed feces from dairy cows fed HiCaCl2, HiMg diets (Fig. 4-3.4). Under waterextraction conditions used in th is experiment (and most e nvironmental conditions) hydroxylapatite is a mineral with greater stability (reduced solubility) than withlockite ( -Tri-Ca/Mg phosphate) (Lindsay, 1979). Mineral sequence docume nted in fecal samples is consistent with the observed differences in WEP from those samples and leads us to believe dietary modifications, particularly increase d dietary Ca availability, affect ed the inorganic form in which P is excreted and ther efore its solubility. There is uncertainty as to whether these minerals were present in feces prior to ashing as they were not detected by X-ray diffraction. We su spect that (a) they may have been present in concentrations below detection, (b) they were no t crystalline and theref ore not detected by XRD or (c) they were not present at all. Despite this uncertainty, there is no doubt fecal samples from cows fed HiCa diets had lower cumulative WEP in correspondence with th e expected solubility of the minerals found in those samples.

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82 Dot maps and elemental spectral data showed high spatial associations between Ca, Mg and P in areas first identified with high P con centrations; in some cases the association was mainly with Mg, or Ca and in other cases with both (Fig 4-4). This tr end was consistent in samples from the two diets analyzed, lowest a nd highest WEP. These findings support the idea of a solid phase where both Ca and Mg are combined with P, independently (P with Ca or Mg) or together (P with both Ca and Mg). Impact of Dietary Modifications on Fecal-P Solubility Dietary Ca concentration affected fecal TP concentration. Cows consuming HiCa diets had lower fecal TP than cows being fed LoCa diet (4.8 vs. 5.3 g P kg-1 feces; P = 0.009). This difference resulted in increased total excretion of P from cows fed LoCa diets (34.5 vs. 30.5 g P cow-1 d-1 feces; P = 0.005). As a result of these differences and the effect of in creasing dietary Ca on fecal WEP, fecal samples from cows under Hi Ca diets had lower cumulative WEP after ten extractions. Increasing dietary Ca concentra tion reduced cumulative WEP (15.9 vs. 11.7 g P cow-1 d-1; P <.0001). Main dietary treatments of Ca source and Mg concentrations had no effect on fecal TP concentration or output (Table 4-5). However, feeding CaCl2 reduced cumulative WEP from feces and daily WEP when fecal excretion was fa ctored in (Table 4-6). We believe feeding CaCl2, a Ca source of greater solubility in the rumen than CaCO3 (100 vs. 68%) provided a more steady flow of Ca to the small intestine, site wh ere Ca and P may first interact. Once in the small intestine food is not subject to acidic pH conditions and therefore any Ca-P formed may be excreted in that condition. Based on XRD data of ashed fecal samples, formation of less soluble Ca-P is expected in the GIT of cows fed higher available Ca, reason for the reduction in cumulative WEP of fecal samples from cows being fed the CaCl2 diets (15.1 vs. 12.5 g WEP cow-1 d-1).

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83 Differences in WEP factored in with fecal out put provide an opportunity to better observe the impact of dietary modifications on the overall P balance in a dairy farm. Increasing available dietary Ca reduced the P solubility a nd its potential for offsite movement. Conclusions Increasing Ca availability in the GIT eith er by increased dietary concentration or by feeding a more soluble Ca form reduced WEP fro m feces, but dietary Mg concentrations did not have an effect on WEP. High correlation coeffici ents in the release of WECa and WEMg with WEP in water extractions suggest Ca, Mg and P are associated in feces. This inference is supported by solid-state evidence of spatial presence of Ca, Mg, and P in fecal samples regardless of the dietary treatment and by XRD results from ashed fecal samples. Phosphorus solubility for dairy cows feces can be reduced by dietary control of Ca and Mg when the same dietary P concentration is fed; the effect is driven by increased di etary Ca availability.

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84Table 4-1. Nutrient content of diets with different Ca and Mg concentrations and two calcium so urces fed to the lactating dair y cows. Ca Source, dietary Ca concentra tion, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 Chemical Composition CP, g kg-1 DM 161 160 161 157 158 158 159 161 ADF, g kg-1 DM 203 202 202 202 204 200 202 199 NDF, g kg-1 DM 354 347 355 344 352 355 354 352 Lignin, g kg-1 DM 35 34 35 34 35 34 35 34 Non-fiber carbo-hydrates, g kg-1 DM 394 398 384 390 393 381 385 365 Ash, g kg-1 DM 57 60 66 74 63 72 68 87 P, g kg-1 DM 3.8 3.8 3.9 3.8 3.8 3.7 3.8 3.7 Ca, g kg-1 DM 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Mg, g kg-1 DM 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 K, g kg-1 DM 12.0 12.0 13.0 13.0 13.0 13.0 12.0 13.0 Na, g kg-1 DM 5.0 4.0 4.0 5.0 4.0 5.0 4.0 5.0 S, g kg-1 DM 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Cl, g kg-1 DM 4.0 4.0 4.0 4.0 7.0 14.0 8.0 12.0 Fe, mg kg-1 DM 160 153 179 186 183 157 168 159 Zn, mg kg-1 DM 137 119 183 154 279 112 144 100 Cu, mg kg-1 DM 41.2 32.9 52.8 51.4 66.4 35.7 40.0 28.1 Mn, mg kg-1 DM 91.7 79.6 120 119 244 82.9 107 70.1 CP= Crude Protein; NDF= Neutral Detergent Fiber; ADF= Acid Detergent Fiber; DM= Dry Matter.

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85Table 4-2. P value for orthogonal single degree of freed om contrast for total dietary concentr ations of phosphorus (P), calcium (Ca), and magnesium. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 TP, g kg-1 0.1345 0.1345 0.5811 1.0000 0.9723 0.6047 0.3075 TCa, g kg-1 0.0244 <.0001 0.0915 0.0473 0.7310 0.0790 0.9838 TMg, g kg-1 0.1291 0.8452 0.0814 <.0001 0.0009 0.2285 0.2444 Table 4-3. Total (T) fecal and water-extractab le (WE) concentrations of P, Ca, and Mg in lactating dairy cows feces after ten successive extractions with water. Ca Source, dietary Ca concentra tion, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 TP, g kg-1 5.3 4.6 5.5 5.2 5.3 4.7 5.0 4.8 TCa, g kg-1 15.6 20.2 13.2 22.8 14.8 23.6 14.9 23.4 TMg, g kg-1 6.1 5.6 9.4 7.9 5.3 5.8 7.5 8.1 WEP, g kg-1 2.8 2.1 2.4 2.0 2.4 1.68 2.3 1.5 WECa, g kg-1 5.0 6.7 4.5 5.5 4.8 6.9 4.8 5.6 WEMg, g kg-1 4.6 4.7 7.8 6.4 3.6 5.2 5.7 5.7 ------------WE mineral : Total Mineral ------------WEP, g 100g-1 53.4 45.2 44.4 37.9 43.7 36.2 47.3 32.3 Ca, g 100g-1 34.0 33.6 33.8 25.0 33.7 31.0 34.5 24.5 Mg, g 100g-1 77.3 82.7 84.2 81.4 70.0 91.4 78.5 69.8

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86Table 4-4. P values for total (T), cumulative wate r-extractable (WE), and wate r-extractable:Total concen tration of phosphorus (P), calcium (Ca), and magnesium (Mg) in feces of lactating dairy cows receiving two Ca sources, two Ca concentrations, and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 TP, g kg-1 0.202 0.009 0.941 0.442 0.147 0.191 0.910 TCa, g kg-1 0.045 <.0001 0.236 0.907 0.938 0.054 0.044 TMg, g kg-1 0.005 0.313 0.001 <.0001 0.175 0.280 0.141 WEP, g kg-1 0.002 <.0001 0.366 0.073 0.436 0.759 0.462 WECa, g kg-1 0.853 <.0001 0.847 0.016 0.735 0.096 0.643 WEMg, g kg-1 0.004 0.738 0.0143 <.0001 0.053 0.009 0.869 ------------WE mineral : Total Mineral ------------P, g 100g-1 0.014 <.0001 0.376 0.053 0.075 0.479 0.295 Ca, g 100g-1 0.684 0.002 0.616 0.036 0.656 0.019 0.887 Mg, g 100g-1 0.194 0.208 0.407 0.612 0.197 0.003 0.099 Table 4-5. Correlation coefficient (r2) between water-extracta ble P (WEP) and water-extractable Ca (WECa), water-extractable Mg (WEMg) or water-extractable Ca+ water-extractable Mg in fe cal samples from lactating dair y cows fed diets differing in Ca source, Ca concentra tion and Mg concentration. Ca Source, dietary Ca concentra tion, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Correlation Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 WEP vs. WECa 0.60 0.43 0.20 0.12 0.46 0.35 0.51 0.11 WEP vs.WEMg 0.81 0.61 0.55 0.65 0.53 0.65 0.67 0.55 WEP vs.WECa + WEMg 0.77 0.61 0.49 0.50 0.55 0.35 0.69 0.45

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87Table 4-6. Least squared means and SEM for fecal output, P concentration in the feces, P excretion via feces and the cumulativ e WEP per kilogram of feces, per day per cow and its implications in a 1000 head farm of lactating dairy cows receiving two Ca sources, two Ca concentrations and two Mg concentrations. Ca Source, dietary Ca concentration, and dietary Mg concentration CaCO3 CaCl2 Dietary Ca (g kg-1 of DM) Dietary Ca (g kg-1 of DM) 6.3 8.6 6.4 9.5 6.5 9.5 6.5 10.3 Measure Dietary Mg (g kg-1 of DM) Dietary Mg (g kg-1 of DM) 2.4 2.4 4.3 4.0 2.6 2.7 3.7 3.8 SEM Phosphorus Excretion Fecal output, kg d-1 cow-1 6.7 6.5 6.3 6.4 6.9 6.3 6.6 6.2 0.2 P conc.in feces, g kg-1 5.3 4.6 5.5 5.2 5.3 4.7 5.0 4.8 0.2 P out in feces, g d-1cow-1 35.5 30.1 34.2 33.5 36.3 28.9 32.1 29.6 1.9 Phosphorus Solubility Cumulative WEP, g kg-1 of feces 2.7 2.2 2.4 2.0 2.3 1.8 2.2 1.3 0.14 Cumulative WEP, g d-1 cow-1 17.9 14.5 15.3 12.7 15.6 11.4 14.7 8.28 0.15 WEP, g 100 g-1 of TP 53.4 45.2 44.4 37.9 43.7 36.2 47.3 32.3 2.9 Impact 1000 head dairy TP Excreted, kg d-1 35.5 30.1 34.2 33.5 36.3 28.9 32.1 29.6 1.9 Cumulative WEP, kg d-1 17.9 14.5 15.3 12.7 15.6 11.4 14.7 8.28 0.15

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88Table 4-7. P values for fecal output, P concentrati on in the feces, P excretion via feces a nd the cumulative water-extractable P per kilogram of feces, per day per cow and its implications in a 1000 head farm of lactating dairy cows receiving two Ca sources, two Ca concentrations and two Mg concentrations. CaCO3 vs. CaCl2 (1: Ca Source) High vs. Low Ca (2: Ca Conc.) Interaction 1 by 2 High vs. Low Mg (3: Mg Conc.) Interaction 1 by 3 Interaction 2 by 3 Three way interaction 1 by 2 by 3 Phosphorus Excretion Fecal output, kg d-1 0.863 0.071 0.176 0.114 0.747 0.391 0.991 P conc.in feces, g kg-1 0.202 0.009 0.941 0.442 0.147 0.191 0.910 P output in feces, g d-1 0.247 0.005 0.505 0.812 0.331 0.076 0.968 Phosphorus Solubility Cumulative WEP, g kg-1 of feces 0.0018 <.0001 0.3657 0.0731 0.4763 0.7593 0.4622 Cumulative WEP, g d-1 cow-1 0.0114 <.0001 0.1910 0.0331 0.6379 0.3684 0.5154 WEP, g 100 g-1 of TP 0.0142 <.0001 0.3756 0.0525 0.0753 0.4789 0.2951

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89 WEP = -566Mg + 2549 r2 = 0.01 CV= 30 WEP = -745Ca + 3565 r2 = 0.28 CV= 26 0 500 1,000 1,500 2,000 2,500 3,000 3,500 0.05.010.015.020.025.030.0 Feces Mg, Ca (g kg-1of DM)WEP (mg kg-1) Mg Ca WEP = -1394Mg + 2599 r2 = 0.03 CV=30 WEP = -2408Ca + 4057 r2 = 0.35 CV= 24 0 500 1,000 1,500 2,000 2,500 3,000 3,500 0.02.04.06.08.010.012.0 Dietary Mg, Ca (g kg-1of DM)WEP (mg kg-1) Mg Caa b WEP = -566Mg + 2549 r2 = 0.01 CV= 30 WEP = -745Ca + 3565 r2 = 0.28 CV= 26 0 500 1,000 1,500 2,000 2,500 3,000 3,500 0.05.010.015.020.025.030.0 Feces Mg, Ca (g kg-1of DM)WEP (mg kg-1) Mg Ca WEP = -1394Mg + 2599 r2 = 0.03 CV=30 WEP = -2408Ca + 4057 r2 = 0.35 CV= 24 0 500 1,000 1,500 2,000 2,500 3,000 3,500 0.02.04.06.08.010.012.0 Dietary Mg, Ca (g kg-1of DM)WEP (mg kg-1) Mg Caa b Figure 4-1. Variation in water extractable P with respect to changes in dietary and fecal concentrations of Mg and Ca.

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90 0 500 1000 1500 2000 2500 3000 012345678910 Extraction Number Ca in Diet (mg kg-1)Water Extractable P (mg kg-1) 6.3 6.4 6.5 6.5 8.6 9.5 9.5 10.3Dietary Ca (g kg-1) 0 1000 2000 3000 4000 5000 6000 7000 8000 012345678910 Extraction NumberWater Extractable Ca (mg kg-1) 6.3 6.4 6.5 6.5 8.6 9.5 9.5 10.3Dietary Ca (g kg-1) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 012345678910 Extraction Number Mg, Ca in Diet (g kg-1)Water Extractable Mg (mg kg-1) 6.3 6.4 6.5 6.5 8.6 9.5 9.5 10.3 Dietary Ca and Mg (g kg-1) 6.3 Ca, 2.4 Mg 6.4 Ca, 4.3 Mg 6.5 Ca, 2.6 Mg 6.5 Ca, 3.7 Mg 8.6 Ca, 2.4 Mg 9.5 Ca, 4.0 Mg 9.5 Ca, 2.7 Mg 10.3 Ca, 3.8 Mg CaCO3 CaCl2 0 500 1000 1500 2000 2500 3000 012345678910 Extraction Number Ca in Diet (mg kg-1)Water Extractable P (mg kg-1) 6.3 6.4 6.5 6.5 8.6 9.5 9.5 10.3Dietary Ca (g kg-1) 0 1000 2000 3000 4000 5000 6000 7000 8000 012345678910 Extraction NumberWater Extractable Ca (mg kg-1) 6.3 6.4 6.5 6.5 8.6 9.5 9.5 10.3Dietary Ca (g kg-1) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 012345678910 Extraction Number Mg, Ca in Diet (g kg-1)Water Extractable Mg (mg kg-1) 6.3 6.4 6.5 6.5 8.6 9.5 9.5 10.3 Dietary Ca and Mg (g kg-1) 6.3 Ca, 2.4 Mg 6.4 Ca, 4.3 Mg 6.5 Ca, 2.6 Mg 6.5 Ca, 3.7 Mg 8.6 Ca, 2.4 Mg 9.5 Ca, 4.0 Mg 9.5 Ca, 2.7 Mg 10.3 Ca, 3.8 Mg CaCO3 CaCl2 Figure 4-2. Cumulative water extrac table P, Ca and Mg with cha nges in dietary concentrations of Ca and Mg.

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91 C W W W S C W W/A W S A A C W W/A W S A A C S A A A W 1 2 3 `4 C W W W S C W W/A W S A A C W W/A W S A A C S A A A W 1 2 3 `4 Figure 4-3. X-ray diffraction analysis of ashed fecal samples of randomly selected dairy cows receiving (1) LoCaCO3, HiMg; (2) LoCaCO3, LoMg; (3) HiCaCO3, HiMg; and (4) HiCaCl2, HiMg. Minerals present are: C: calcite (CaCO3); S: silvite (KCl); W: whitlockite (Ca,Mg)3(PO4)2; and A: hydroxyl-apatite Ca5(PO4)3(OH, Cl, F). LoCa refers to low dietary Ca concentration wher eas HiCa is high dietary Ca concentration; same applies to Mg. Each line within a graph represents a different fecal sample.

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92 Figure 4-4. Dot maps and P, Ca and Mg elem ental spectra from areas identified with high P concentrations of randomly selected fecal samples of cows being fed low Ca and high Mg concentrations or high Ca and hi gh Mg concentration in their diets.

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93 CHAPTER 5 DIETARY CALCIUM AND MAGNESIUM EFFECT ON PHOSPHORUS SOLUBILITY OF DAIRY FECES IN THREE DIFFERENT SOILS; AN INCUBATION STUDY Introduction Animal waste from dairy farms is commonly a pplied to soils as manure (mixtures of feces, bedding materials, water, etc.). Farm management strategies often include areas of animal rest where considerable amounts of feces can accumu late on the soil surface. Differences in P solubility and chemical forms between manure (m ix of feces and urine) and feces have been documented (Toor et al., 2005), but risk assessmen t tools (P Indexes) make no distinction for P application based on -dairywaste ch aracteristics. It is assumed that most P is highly soluble or becomes plant available soon after application (Cooperba nd and Good, 2002). Surface soils where dairy manure accumulates over long period s of time are a source of P for offsite movement (Sharpley et al., 2001, Sims et al., 1998) This P reaches water bodies and promotes eutrophication, negatively impacting water quality (Ebeling et al., 2002). Research efforts on controlling P balances at the farm level have focused on understanding P movement (Beauchemin et al., 1998, Turner and Haygarth, 2000), soil P retention capacity (Khiari et al., 2000, Rhue et al ., 2006), reducing P concentrations in the feed to minimize P excretion (Dou et al., 2003, Wu and Satter, 2000), or evaluati ng manure chemical and physical characteristics, with (Dou et al., 2002) or w ithout (He et al., 2004) diet ary control. However, little emphasis has been placed on dietary modificatio ns to increase long term P stability in soils where feces-derived components can play a major role in ultimate P solubility. Cooperband and Good (2002) showed in incu bation experiments of poultry manure with soil that sparingly solu ble Ca and Mg phosphates minerals c ontrol soil solution P concentrations. Dairy manure impacted soils, on the other hand ofte n have conditions associated with increased P stability by its association with Ca (high pH and elevated concen trations of P and Ca) yet solid

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94 state crystalline phosphate minerals have not been id entified in these soils (Harris et al., 1994) or in (dairy) manure-soil incubations (Cooperba nd and Good, 2002). Instead, Josan et al. (2005) and Nair et al. (1995) demonstr ated that P remains highly so luble, even after years of abandonment. Elevated concentrations of Mg a nd dissolved organic carbon have been proposed as inhibitors of more stable calcium-phosphate minerals (Ca-P) in manure amended soils (Harris et al., 1994; Josan et al., 2005). The objective of this study was to test the following hypotheses: (i) Increased Ca availability in the GIT of lactating dairy cows will favor Ca-P formation in feces therefore reduce P solubility in soil-feces incubations, and (ii) reduction in P solubility with time for soilfeces mixtures is favored by higher available Ca in the diet. The approach was to evaluate effects of dietary Ca and Mg concentrations a nd of solubility of Ca source on P concentration and solubility in soils incubated with feces from lactating cows. Materials and Methods Selected dairy fecal samples were incubated with the surface horizon (Ap) of three soil orders (Table 5.1) in a laboratory setting. Dry feces were added to each soil to supply a TP concentration of 1000 mg P kg-1, rate in the low range of typical heavy intensity areas in dairy farms. Samples were kept under controlled cond itions at room temperature for 6 months. Moisture content was checked every 2 weeks a nd maintained at 30% by weight. Consecutive water extractions at a ratio of 1: 100 feces:water were used as a i ndication of long term solubility of P in the incubations. Soils from the three dominant soil orders of Florida (Spodosol, Entisol, and Ultisol) were collected. The soils classified at the subgroup level as Alaquod, Quartzipsamment, and Paleudult, respectively. Soil morphological, physical, and chemical characteristics (Tables 5.1 and 5.2) were reasonably representative of these suborde rs as they occur in Florida based on comparison

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95 with data collected under the Florida Soil Survey Program. They were collected from forested areas in Alachua and Levy Counties, Florida, where recent agricultural impact was minimal. None were elevated in P. Content of clay, Al, and Fe followed a typical trend among these soils: Spodosol
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96 Philadelphia, PA). Measured concentrations of P, Ca and Mg in fecal samples varied with respect to formulations (Table 5.3); however, these differences did not alter the obje ctives of the study. Composited fecal samples from 4 cows (replicati ons) from each of four treatments (out of eight) were selected to incubate with soils. Trea tments selected represent both concentrations of Ca and Mg and two sources of Ca. Treatments c hosen differed in WEP but did not represent the extremes (highest and lowest WEP). To minimi ze effects from differences in organic matter application, individual fecal samples chosen had a TP con centration between 4500 to 5500 mg kg-1 (Table 5-3). Samples from animals receiving high diet Ca concentrations are referred as HiCa whereas HiMg is used for high dietary Mg. Fecal samples (treatments) selected were from animals being fed: (i) LoCaCO3, HiMg; (ii) HiCaCO3, HiMg; (iii) LoCaCl2, LoMg; (iv) HiCaCl2, LoMg. Mixture of incubated soils and fecal samples will be discussed on the basis of the Ca source and concentration of Ca and Mg used in the feeding experiment; for example, animals receiving CaCO3 at 6.4 g Ca kg-1 and 4.3 g Mg kg-1 of dietary DM will be referred as LoCaCO3HiMg. Successive water extractions at room temperature (~25 C) were performed for all soilfeces mixes after 1, 25, and 42 weeks of incubations at a 100:1 water:soil+feces ratio. Incubated samples were air dried prior to extraction. After 1 h of shaking time, samples were centrifuged at 4000x g for 5 minutes. Samples incubated for 25 week s were consecutively extracted seven more times for a total of eight extr actions. Supernatants were filt ered (0.45 m) and analyzed for extractable phosphorus (WEP), Ca (WECa), and Mg WEMg. Water-extractable Ca and Mg were measured by atomic absorption spectroscopy; P was determined on a UV-visible recording

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97 spectrophotometer at 880 nm wave-length via the molybdate-blue colorimetric method (Murphy and Riley, 1962) (U.S. EPA, 1993; method 365.1). Two samples from each of two treatments (highe st and lowest WEP) were analyzed by xray diffraction (XRD). Samples were first sieved to pass a 53 m screen, then clayand silt size fractions were mounted on a porous ceramic tile for the XRD scan. Statistical Analyses: The statistical model used was a 3 x 4 factorial design with treatment (four diet), soils (three soils), a nd replications (four per treatmen t). Single data measurements of Ca, Mg and P were analyzed using the GLM procedure of SAS whereas the Mixed Linear Model was used for repeated measures from consecuti ve extractions of the same element; the slice option was used to detect differences in i ndividual extractions. Ort hogonal single degree of freedom contrasts and Tukey-Kramer test were us ed to detect differences among main treatment effects and least squared means of individual treatmen ts, respectively. Corre lations and nonlinear regressions were used to descri be relationships between elements in our analysis. Differences discussed in the text were significant at P 0.05 unless otherwise indicated. Results and Discussion Differences in chemical and physical charac teristics of the thr ee soils used in the incubations (Table 5-2) are c onsistent with WEP results. Th e A horizon of Spodosol had the lowest concentration of P and cations (Ca, M g, Fe and Al), both as total and Mehlich-1 extractable, and the lowest pH, organic matter and clay content. Extreme depletion of metals and clay is characteristics of Spodosol surface horizon s due to the intense podzolization that defines soils of the Spodosol order. The Spodosol A horiz on also had the lowest P retention capacity (Table 5-2), which is also typical (Nair et al., 1999, Yuan and Lavkulich, 1994) due to the lack of P-retaining components (clay-sized metal oxides) and therefore the highest WEP at all times (Fig. 5-1) regardless of treatment when compared with its counterpart in either of the other two

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98 soils used. The A horizon of the Ultisol, on th e other hand, had the high est concentration of metals and clay and the highest P retention ca pacity, consistent with the tendency towards a reduction in WEP over time (25 and 42 weeks). Wa ter-extractable P after 25 and 43 weeks from feces incubated with the Entisol s A horizon showed an intermediate P release behavior which would be expected from its interm ediate properties (metal and clay content; P retention capacity) relative to the other 2 soils. Because of the soil by time interaction (Table 5. 4) it was not possible to test the effect of incubation time on WEP; however, it is apparent that there was no reduc tion of WEP during the 42 weeks (Fig. 1). In effect, hypothesis (ii) that high available Ca in the diet would result in greater P stabilization over time was not supported for the 42 week period. Interaction of time by soil affected the overa ll WEP (average of all diets within time) (Table 5.4). This interaction occurred between sa mples incubated with the Ultisol and Entisol. At time zero the Ultisol had higher WEP across diets than the Entisol (263.5 vs. 247.8 mg kg-1), but after 6 months of incubation WEP from the Ultisol was lower than the Entisol (260.5 vs. 300.7 mg kg-1). Samples incubated with Spodosol had hi gher WEP vales at all times (Fig. 5-1). Effect of dietary treatments on WEP from f ecal samples observed in chapter 4 was also documented in feces-soil incubations. Increase d dietary Ca concentration from 6.4 to 9.5 g kg-1of diet DM reduced WEP in feces-soil incubations (369 vs 228 g kg-1 of mix) (Fig. 5-2), supporting hypothesis (i). This represents a reduction in WEP based on dietary modifications from 35.1 % to 21.7% of the TP in the soil-feces mix; alt hough these values are poole d means across soils the same trend was documented in each soil type at all times (no soil by diet interaction). The reduction in WEP from fecal samples of anim als receiving higher dietary Ca suggests the

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99 formation of a more stable (i.e. less soluble) fo rm of P that likely formed in the GIT of the animal. Chemical conditions in the animals GIT be fore the small intestine do not favor the formation of such a phase. In the fore-stomach (reticulum-rumen) of lactating dairy cows, under most circumstances, pH ranges from 5.8 to 6.5; with 5.8 considered desirable as this is the threshold point for subclinical ruminal acidosis (Yang et al., 2 001). A pH of 6.5 favors formation of Ca-P, but nutrients remain in the reticul o-rumen only temporarily, then undergo an acidic stage at the abomasum (true-stomach) where the pH can be below 2.0 due to the release of HCl (Barry and Manley, 1984). The acidity of the f eed (known as chyme) when it enters the small intestine stimulates secretions from the pancreas These secretions contain bicarbonate, intended to neutralize HCl present in the chyme. At this point (or thereafter) is where a stable P compound can form and ultimately be excreted. Given fecal sample handling and feces-soil mixes used in this experiment, P solubility reduction in manure-impacted soils was achieved preemptively via dietary control; Ca concentration was an effectiv e dietary factor when cows were fed at the recommended dietary P concentration (NRC, 2001). There was an effect of soil and diet on cumu lative WEP after eight consecutive extractions from fecal samples after 25 weeks of incubation at a 1:100 feces-soil mix to water ratio (Table 5.6); interaction between soil a nd diet did not affect cumula tive WEP. Dietary effect was consistent with that observed for fecal samples and for feces-soil mix single extractions at 1, 25 and 42 weeks on WEP. There was no effect of dietary Ca source or the interaction of dietary Ca source by dietary Ca concentration. Dietary Ca concentration affected WEP; HiCa diets (in c ontrast to LoCa diets) reduced WEP from fecal samples incubated with the Ap horizon of both Entisol and Spodosol;

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100 however, there was no dietary Ca concentration effect on WEP from fecal samples incubated with the Ap horizon of Ultisol (T able 5.8) because of the greater P retention capacity present in this soil, therefore reducing both the overall P solubility and the treatment effects. Fecal samples incubated with Ap horizon from Ultisol showed the lowest cumulative WEP concentrations (Fig. 5.3), behavior in agreement with the expected P retention capac ity (relative to the other two soils) based on physical and chemical characteristic s of the Ultisol, particularly soil texture and total and Mehlich-1 extractable conc entrations of Al (Table 5.2). Th ese findings led us to believe soil solution P in Ultisol is interacting with the soils active sites fo r P retention. Therefore solution concentrations are being controlled by sorption-desorption mechanisms, particularly P associated with amorphous Fe and Al forms. These P fixing mechanism may be the reason for the reduction in WEP with time, as observed by Sartain (1980) who documented a reduction in extractable P with multiple extractants; during his experiment, soil TP concentration remained constant. Cooperband and Good (2002) also prop osed sorption-desorption as the processes controlling P solubility from da iry-manure amended Mollisols. The Entisol and Spodosol showed increased cu mulative WEP over eight extractions (Table 5-8). This observation agrees with soil characteri stics (Table 5.2). In soils with low P retention capacity, like the Entisol and Spodosol in this experiment, fecal (manure) components and characteristics are expected to play a major role in the overall soil P release. Increasing dietary Ca concentration from 6.5 to 9.5 g Ca kg-1 dietary DM effectively reduced WEP from feces incubated with Entisol (834.0 vs. 634.5 g WEP kg-1 soil) and Spodosol (1014.1 vs. 774.7 g WEP kg-1 soil) (Table 5-7). This treatment effect on cumulative WEP after 25 weeks of incubation is similar to those observed in singl e water extractions at three diffe rent times of incubation (Fig 52).

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101 Dairy-manure impacted soils have been shown to continue releasing high P concentrations (up to 100 mg P kg-1 soil; 1:10 soil to water extractions) even after years of abandonment (Josan et al., 2005). After fractionation ex tractions it has been shown that P in the A horizon is mainly associated with Ca and Mg (Nai r et al., 1995) and it has been propos ed that sparingly-soluble Caand/or Mg-P phases may control rele ase of P (Josan et al., 2005). This idea is consistent with the high correlation observed between th e release of Ca and Mg (indivi dually or collectively) with that of P from consecutive extr actions (Fig. 5-3) and with xray diffraction results from ashed fecal samples where crystalline forms of Ca and Mg were found; a more stable Ca-P -apatitewas present in feces with lower WEP concentra tions whereas a more soluble Ca(Mg)-P form withlockitewas found in feces w ith higher WEP. As discussed in chapter 4, these P forms do not necessarily exist in fresh or dried fecal sample s, but it is likely that their stoichiometric precursors that reflect their relative solubility are present in the feces. These changes in P chemical composition and structural arrangement are likely the result of dietary modifications, particularly increased Ca concentration, and the main reason for the differences in WEP in soilfeces incubations (Table 5-7). Cumulative release of WECa and WEMg were a ssociated with WEP (Fig 5-3). Associated release differed with changes in di et and soils; however, correlati ons were significant in all cases when analyzed within soil and diet. Co rrelation of cumulative WEP with cumulative WECa+WEMg from fecal samples incubated with the Spodosol showed the influence of dietary modifications. The main difference, as discus sed above, was between Lo and HiCa diets; however, the effect of Mg was more clearly obs erved in this correlati on. When diets contained HiCa concentrations, addition of Mg (HiMg) did not make a difference, but when diets contained LoCa concentrations, increasing dietary Mg increased WE concentrations of both P and the

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102 combination of Ca and Mg (Fig. 5-3). This sugges ts a higher association of P with Mg, resulting in their higher WE concentration. This diet ary effect was not obser ved in fecal samples incubated with the Ultisol and Entisol because of their higher P retention capacity. Conclusions Increasing Ca concentration in the diet of lactating dairy cows from 6.5 to 9.5 g Ca kg-1 dietary DM preemptively reduced P release from incubated feces-soil mixtures. This effect was most pronounced in Spodosol, probably because it had minimal components to retain the P released from the feces. Soil characteristics shou ld be taken into account when considering P loading rates; soils used in th is experiment differed in WEP c oncentrations. Changes in dietary Ca source and Mg concentrations had no effect on WEP. No further P stabil ization effect of high available dietary Ca was observe d during a 42 week period. This la ck of a time effect suggests that Ca, Mg, and P interactions in the GIT ma y be the major determinant of the subsequent environmental fate of fecal P. However, a l onger incubation time may ultimately confirm a greater stabilization with time for the high Ca diets.

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103 Table 5-1. Morphological summary of soils from which surface horizon samples were collected for the incubation experiment. Horizon Lower Depth Texture Color Ultisol Hue Value Chroma Ap 20 Sand 10YR 4 2 A/E 38 Sand 10YR 4/5 2/3 E 51 Sand 10YR 5 4 Bt1 63 Loamy Sand 10YR 5 6 Bt2 92 Sandy Loam 10YR 5 6 Btg 110+ Sandy Clay Loam 10YR 6 2 Spodosol Ap 8 Sand 10YR 4 1 E1 20 Sand 10YR 5 1 E2 52 Sand 10YR 7 1 Bh1 59 Sand 10YR 2 1 Bh2 66 Sand 10YR 3 2 Bw 134 Sand 7.5YR 4 3 Btg 144+ Sandy Clay Loam 10YR 6 2 Entisol Ap 6 Sand 10YR 4 2 E1 50 Sand 10YR 5 4 E2 100+ Sand 10YR 6 6 Table 5-2. Chemical and physical characteristics of th e surface horizon (Ap) from each soil type used for the incubation. Ultisol Spodosol Entisol Total (mg kg-1) P 46.2 8.2 133.3 Ca 191.2 130.0 271.3 Mg 106.3 10.6 87.2 Fe 851.3 124.9 796.9 Al 4877 1210 2490 Mehlich-1 (mg kg-1) P 2.0 1.5 6.9 Ca 95.8 47.4 202.4 Mg 19.8 6.4 25.7 Fe 15.2 5.9 21.3 Al 371.4 38.1 145.3 RPA 91 11 66 pH 4.3 4.2 5.3 Organic Matter (g kg-1) 40.9 8.0 21.6 Particle Size Dist. Sand (g kg-1) 931 969 979 Silt (g kg-1) 46 25 8 Clay (g kg-1) 23 6 13 : Relative P absorption as determined by single-point P absorption isotherm as measured in the A horizon of each soil order collected.

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104 Table 5-3. Chemical characterist ics of fecal samples used for th e incubation with three different soils orders. Ca Source CaCO3 CaCl2 Dietary Ca (g kg-1 DM) 6.4 9.5 6.5 9.5 Dietary Mg (g kg-1 DM) 4.3 4.0 2.7 2.7 Mineral --------------mg kg-1 --------------P 5170 380 5012 354 4940 508 4795 308 Mg 8925 430 7980 733 4894 490 5958 1068 Ca 12432 1027 23539 2004 12633 1249 24894 4025 Cumulative WEP* 2370 225 1931 356 2364 568 1808 484 *: Cumulative WEP after 10 successive extractions using a 1:100 feces to water ratio. Table 5-4. ANOVA table of waterextractable P (WEP) from soilfeces at different times of incubation (1, 25, and 42 weeks). Source DF Type I SS Mean Square F Value Pr > F Replication 3 99092 33030 8.65 0.0134 Soil 2 366575 183287 48.0 0.0002 Error 1 6 22914 3819 --Diet 3 760066 253355 71.2 <.0001 Soil x Diet Int. 6 19064 3177 0.89 0.5138 Error 2 27 96054 3557 --Time 2 40738 20369 5.59 0.0055 Soil x Time Int. 4 80755 20188 5.54 0.0006 Diet x Time Int. 6 26042 4340 1.19 0.3204 Soil x Diet x Time Int. 12 31129 2594 0.71 0.7345 Error 3 72 262164 3641 --Corrected Total 143 1804600 Error term 1 = Rep x Soil Interaction, Error term 2 = Rep x Soil x Diet Interaction, Error term 3 = Rep x Soil x Diet x Time Interaction.

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105 Table 5-5. Water extractable P fr om a single extraction at 1:100 soil -feces mix to water ratio at different times of incubation and the respect ive standard error of the mean (SEM). Ca Source CaCO3 CaCl2 Dietary Ca (g kg-1 DM) 6.4 9.5 6.5 9.5 Dietary Mg (g kg-1 DM) 4.3 4.0 2.7 2.7 SEM ----Water Extractable P (mg kg-1) ----One week Ultisol 287.7 211.9 351.0 203.3 16.2 Spodosol 357.5 275.8 431.6 246.6 18.9 Entisol 260.6 208.2 325.0 197.3 16.1 25 weeks Ultisol 302.5 211.7 325.4 202.3 22.2 Spodosol 446.8 339.8 494.7 320.6 42.0 Entisol 345.2 198.3 435.8 223.5 36.1 42 weeks Ultisol 290.8 142.7 260.3 119.7 10.5 Spodosol 393.9 294.3 510.0 266.3 46.1 Entisol 412.7 242.8 408.4 205.4 28.7 Table 5-6. ANOVA table of cumulative water-extrac table P (WEP) after eight extractions of at 1:100 soil-feces to water ratio after 25 weeks of incubation. Source DF Type I SS Mean Square F Value Pr > F Soil 2 448509 224255 13.4 <.0001 Diet 3 358381 119460 7.14 0.0008 Soil x Diet Interaction 6 92315 15386 0.92 0.4935 Replication 3 134878 44959 2.69 0.0624 Error 33 552186 16733 Corrected Total 47 1586270 Table 5-7. Cumulative water extractable P (WEP ) from ten consecutive extractions at 1:100 soil-feces mix to water ratio after six months of incubation and the respective standard error of the mean (SEM). Ca Source CaCO3 CaCl2 Dietary Ca (g kg-1 DM) 6.4 9.5 6.5 9.5 Dietary Mg (g kg-1 DM) 4.3 4.0 2.7 2.7 SEM ----Water Extractable P (mg kg-1) ----Time = 25 weeks Ultisol 698.5a 464.5a 701.5a 606.75a 66.5 Spodosol 1040.8a 813.3b 987.5a 736.0b 83.4 Entisol 786.0a 624.8b 882.0a 643.5b 54.2 For each property, mean values within a row follo wed by a different letter were significantly different ( P < 0.05) as determined by Duncan s mean separation procedure.

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106 Table 5-8. P values of water extractable P (WEP) fo r single degree of freedom contrasts based on dietary variables from where fecal samples were collected. High vs. Low Ca CaCO3 vs. CaCl2 Ca Source by Ca Conc. Interaction -------------P values -------------Time = 25 weeks Ultisol 0.2213 0.6268 0.7107 Spodosol 0.0079 0.4587 0.8687 Entisol 0.061 0.8183 0.5081

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107 0 50 100 150 200 250 300 350 400 450 12542 Weeks of IncubationWEP (mg kg-1 of feces-soil mix) Ultisol Spodosol Entisol Figure 5-1. Interaction effect of soil type and time on water-extractable P (WEP) single extractions at 1:100 water to soil-feces in cubations. Data points are averaged across diets. 0 100 200 300 400 500 600 LoCaCO3, HiMgHiCaCO3, HiMgLoCaCl2, LoMgHiCaCl2, LoMg Dietary Ca Source, Ca and Mg ConcentrationWEP (mg kg-1 of feces-soil mix)A A B B LoCaCO3, HiMgHioCaCO3, HiMgLoCaCl2, LoMgHioCaCl2, LoMg 0 100 200 300 400 500 600 LoCaCO3, HiMgHiCaCO3, HiMgLoCaCl2, LoMgHiCaCl2, LoMg Dietary Ca Source, Ca and Mg ConcentrationWEP (mg kg-1 of feces-soil mix)A A B B LoCaCO3, HiMgHioCaCO3, HiMgLoCaCl2, LoMgHioCaCl2, LoMg Figure 5-2. Averaged water-extractable P (WEP) at three different times (1, 25, and 42 weeks) from single extractions at 1:100 ratio of water to soil-feces incubations. LoCaCO3, HiMg HiCaCO3, HiMg LoCaCl2, LoMg HiCaCl2, LoMg

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108 0 5 10 15 20 25 30 35 40 020406080100120140 WECa+WEMg (mmol kg-1)WEP (mmol kg-1) LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMg 0 5 10 15 20 25 30 35 40 020406080100120140 WECa+WEMg (mmol kg-1)WEP (mmol kg-1) LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMga b 0 5 10 15 20 25 30 35 40 020406080100120140 WECa+WEMg (mmol kg-1)WEP (mmol kg-1) LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMg LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMgc 0 5 10 15 20 25 30 35 40 020406080100120140 WECa+WEMg (mmol kg-1)WEP (mmol kg-1) LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMg 0 5 10 15 20 25 30 35 40 020406080100120140 WECa+WEMg (mmol kg-1)WEP (mmol kg-1) LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMga b 0 5 10 15 20 25 30 35 40 020406080100120140 WECa+WEMg (mmol kg-1)WEP (mmol kg-1) LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMg LoCaCO3-HiMg LoCaCl2-LoMg HiCaCO3-HiMg HiCaCl2-LoMgc Figure 5-3. Relationship between water-extract able P (WEP) with WECa+WEMg from eight consecutive extractions at 1:100 soil-feces to water ratio. Fecal samples were incubated for 25 weeks with a) Ultisol, b) Entisol, and c) Spodosol. Data points shown are average of four repl ications standard error.

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109 CHAPTER 6 SUMMMARY AND CONCLUSIONS Dairy manure is a resource used by farmers to satisfy crop nutrient demands. Application of manure at rates to meet plant N requirements results in excess P with respect to plant needs. Over time, this excess P builds up in the soil a nd becomes more susceptible to offsite movement, thereby posing a potential eutrophication risk to water bodies. These risks are directly proportional to the solubility of manure P a nd soils P retention capaci ty. This dissertation addressed the efficacy of dietary manipulations that would reduce P solubility in dairy manure by means other than dietary P reductions alone. It documented effects of dietary Ca and Mg on solubility of P in dairy feces and soil-feces mi xtures incubated over time, and on animal health and performance, and probed physiological and chemical mechanisms responsible for those effects. Reducing dietary P concentrati on is a starting point to optim ize P balance in dairy farms and minimize the potential for negative environmental impact of accumulated P; however, feeding P at the NRC recommended dietary concentration (3.7 g P kg-1 diet DM for lactating dairy cows) is the most viable and safe altern ative to ensure adequate animal productivity and reproductive performance. Excess dietary P has been shown to increase manure TP, particularly the most soluble fraction. In soils with low P retention capacity, manure components (Ca, Mg, dissolved organic carbon) determine P solubility and stabilization in soils; therefore, increasing available Ca concentration in the diet of lact ating dairy cows seems like an alternative to promote formation of stable Ca-P, as opposed to less stable Mg-P or Ca, Mg-P forms, and hence reduce manure-P solubility in soils. Large dietary intakes of Ca and Mg can re duce P absorption by the animal due to the formation of insoluble phospha tes inside the gastro intest inal tract; however, from an

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110 environmental perspective; howev er, their interaction with P ma y decrease P solubility in dairy feces and minimize losses from dairy manure-impact ed soils. Dietary manipulations to increase dietary Ca (and Mg) with an environmental obj ective should take into account animal nutrient requirements and possible detrimental effects of such dietary changes on animal health and performance. Total P fecal concentrations did not relate to P extracted after 10 consecutive water extractions in fecal samples from animals in di fferent physiological states. Calcium and Mg in feces seem to play an important role in contro lling WEP. Increased ratio of P:(Ca+Mg) in feces corresponded to increased WEP. Animals in differe nt physiological states and fed different diets produced feces with different P release characteris tics; overfeeding P to heifers resulted in fecal samples with the greatest cumulative WEP concentrations but not the highest fecal TP concentration. The proportion of TP solubilized after ten consecutive extractions went from ~42% for adult lactating cows fed diets matc hing their P requirements to ~86% for growing heifers fed a diet containing P at 140% of their requirement. Results of this study indicate that physiological state of the animal, dietary P concen tration with respect to animal requirements, and dietary and fecal concentrations of Ca and Mg influence P solubility in feces of Holstein dairy cattle. Use of two Ca sources and two c oncentrations of Ca and Mg in the diet of lactating dairy cows had no effect on milk yield, milk effici ency, fat corrected milk, fat corrected milk efficiency, digestibility of nutrients and overall P balance of lactating dairy cows. There was an effect on milk fat production (% and kg d-1) and milk protein concentration (%). Addition of CaCl2 to rations of lactating dairy cows warrants long term studies because of the tendency to decrease DMI and associated imp lications observed in this study.

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111 Main dietary treatments of Ca source, Ca concentration and Mg concentration had no effect on fecal P concentrations. Increased Ca co ncentration in both diet and feces reduced water extractable P (WEP) from fecal samples. Fecal samples with higher Ca and Mg concentrations showed reduced WEP; possibly, Ca and Mg mutu ally suppressed dissolution of Ca,Mg-P forms by the common ion effect. Increasing Ca activity in the GIT either by in creased concentration or by feeding a more soluble Ca form reduced WEP from feces, but di etary Mg concentrations did not affect WEP. Increased fecal concentrations of Mg and Ca in feces mutually suppressed the dissolution of each element, suggesting that Ca, Mg and P are asso ciated in feces. This is supported by (i) consecutive extraction data; (ii) SEM/EDS anal ysis where close association between Ca, Mg, and P in fecal samples regardless of the diet ary treatment were documented; and (iii) XRD results from ashed fecal samples where hydroxyapatite (HAP), HAP plus Ca,Mg-P (Whitlockite), and Ca,Mg-P are the P forms for hi gh, intermediate, and low dietary available Ca, respectively. Phosphorus solubility in dairy cow feces can be reduced by dietary control of Ca and Mg when the same dietary P concentration is fe d; the effect is driven by increased dietary Ca availability. Inhibitory effect of Mg on formati on of stable Ca-P can be preempted by sufficient available dietary Ca. Increasing Ca concentration in the diet of lactating dairy cows from 6.5 to 9.5 g Ca kg-1 dietary DM preemptively reduced P release from incubated feces-soil mixtures. This effect was most pronounced in the Spodosol, probably because lack of P sorbing co mponents in this soil resulted in almost exclusive control of P re lease by fecal components (as product of dietary treatments). Soil characteristics should be taken into account when considering P loading rates; soils used in this experiment differed in WEP concentrations. Changes in dietary Ca source and

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112 Mg concentrations had no effect on WEP from feces and soil feces incubations. No further P stabilization effect of high avai lable dietary Ca was observed over a 42 week period. This lack of a time effect suggests that Ca, Mg, and P interac tions in the GIT may be the major determinant of the subsequent environmental fate of f ecal P. However, a longer incubation time may ultimately confirm a greater stabilization over time for the high Ca diets.

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122 BIOGRAPHICAL SKETCH Daniel Herrera was born in San Jose, Co sta Rica in 1976. He is the youngest son of Guillermo Herrera and Betty Duran. Daniel went to EARTH University in Costa Rica for his undergraduate education; there he received his B.S in agronomy. His M.Sc. degree is in soil science from the University of Florida wh ere he is now completing his Ph.D. program.